DE69130668T2 - CHEMOLUMINESCENT 3- (SUBSTITUTED ADAMANT-2'-YLIDENE) 1,2-DIOXETANE - Google Patents

Abstract

Enzymatically cleavable chemiluminescent 1,2-dioxetane compounds capable of producing light energy when decomposed, substantially stable at room temperature before a bond by which an enzymatically cleavable labile substituent thereof is intentionally cleaved, are disclosed. These compounds can be represented by formula (I) wherein X and X<1> each represent, individually, hydrogen, a hydroxyl group, a halo substituent, an unsubstituted lower alkyl group, a hydroxy (lower) alkyl group, a halo (lower) alkyl group, a phenyl group, a halophenyl group, an alkoxyphenyl group, a hydroxyalkoxy group, a cyano group, a carboxyl or substituted carboxyl group or an amide group, with at least one of X and X<1> being other than hydrogen; and R1 and R2 individually or together, represent an organic substituent that does not interfere with the production of light when the dioxetane compound is enzymatically cleaved and that satisfies the valence of the dioxetane compound's 4-carbon atom, with the provisos that if R1 and R2 represent individual substituents the R2 substituent is aromatic, heteroaromatic, or an unsaturated substituent in conjugation with an aromatic ring, and that at least one of R1 and R2 is, or R1 and R2 taken together are, an enzymatically cleavable labile group-substituted fluorescent chromophore group that produces a luminescent substance when the enzymatically cleavable labile substituent thereof is removed by an enzyme. The corresponding dioxetanes which, instead of being substituted at the 5' or 7', or at the 5' and 7' positions, instead contain a 4' methylene group, are also disclosed, as are intermediates for all these 3-substituted adamant-2'-ylidenedioxetanes, and their use as reporter molecules in assays.

Description

The invention relates to improved chemiluminescent 1,2-dioxetane compounds. The invention relates in particular to improved enzymatically cleavable chemiluminescent 1,2-dioxetane compounds which contain enzymatically removable labile groups. Such labile groups prevent the molecule from decomposing to generate light, for example visible light or light detectable by suitable instruments, until a suitable enzyme is added to remove the labile group.

One enzyme molecule effects the removal, through a catalytic cycle, of its complementary labile groups from thousands of enzymatically cleavable chemiluminescent 1,2-dioxetane molecules. This is in marked contrast to the situation with chemically cleavable chemiluminescent 1,2-dioxetanes, where one molecule of chemical cleaving agent is required to remove complementary labile groups from each dioxetane molecule. For example, one mole of sodium hydroxide is required to cleave one mole of hydrogen ion from the hydroxyl substituent of the phenyl group in 3-(2'-spiroadamantane)-4-methoxy-4-(3"- hydroxy)phenyl-1,2-dioxetane, whereas only a single mole of alkaline phosphatase ("AP") is required to cleave the phosphoryloxy group in 1,000-5,000 moles of 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane disodium salt per second; see Jablonski, "DNA Probes for Infectious Diseases" (Boca Raton, Fla.: CRC Press, 1989), p. 22.

Enzymatically cleavable light-generating 1,2-dioxetane compounds will usually also contain stabilizing groups such as an adamantylidene group spiro-bonded to the 3-carbon atom of the dioxetane ring, which prevents the dioxetane compound from undergoing significant decomposition at room temperature (about 25ºC) before the bond by which the enzymatically cleavable labile group is bound to the rest of the molecule is intentionally cleaved. The concept of using a spiro-adamantyl group for stability was introduced into 1,2-dioxetane chemistry by Wierynga, et al., Tetrahedron Letters, 169 (1972) and McCapra, et al.; J. Chem. Soc., Chem. Comm.; 944 (1977). These stabilizing groups allow such dioxetanes to be stored for reasonably long periods before use, for example from about 12 months to as long as about 12 years at temperatures in the range of about 4 to about 30°C without substantial decomposition occurring.

The invention further relates to the incorporation of the dioxetane molecules into recognized immunoassays, chemical assays, and nucleic acid probe assays and their use as direct chemical/physical probes for the investigation of the molecular structures or microstructures of various macromolecules, synthetic polymers, proteins, nucleic acids, catalytic antibodies and the like and enables the identification or quantitative Determination of an analyte - the chemical or biological substance whose presence, amount, or structure is to be determined.

State of the art

Chemiluminescent 1,2-dioxetanes have already found increasing importance in recent years, especially with the advancement of enzymatically cleavable chemiluminescent 1,2-dioxetanes as described in WO 88/00695, US 5 089 630, WO 89/06226 and US 4 952 707.

In marked contrast to the enzymatically cleavable 1,2-dioxetanes, the various chemically cleavable chemiluminescent 1,2-dioxetanes known to date have found little, if any, use as reporter molecules in any type of analytical procedure, and certainly not in bioassays. This is because the known chemically cleavable compounds are for the most part water insoluble - except for certain acetoxy-substituted 1,2-dioxetanes, which are somewhat water soluble as well as soluble in organic solvents, and are therefore not useful in biological assays unless they are slightly modified by the addition of groups or substituents that allow conjugation to a biological species, such as an antibody, whereby such conjugated chemically cleavable 1,2-dioxetanes can be used as chemically activated chemiluminogenic labels.

The water solubility of typical enzymatically cleavable chemiluminescent 1,2- dioxetanes, on the other hand - for example, adamantyl-linked enzymatically cleavable 1,2- dioxetanes which decompose in the presence of a suitable enzyme with light emission - such as 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo[3.3.1.13,7]decan]-4-yl)phenyl phosphate and its salts, for example disodium salt, as identified hereinafter briefly as adamantylidenemethoxyphenoxyphosphorylated dioxetane ("AMPPD") and 3-(4-methoxyspiro[1,2-dioxetane-3,2'- tricyclo[3.3.1.13,7]decan]-4-yl)phenyloxy-3"-β-D-galactopyranoside and its salts ("AMPGD") - make them ideally suited for use as reporter molecules in many types of analytical procedures carried out in aqueous media and particularly in bioassays.

It has been observed that in aqueous solution and also in the presence of chemiluminescent enhancers, for example a polymeric ammonium, phosphonium or sulfonium salt such as poly[vinylbenzyl(benzyldimethylammonium chloride)] ("BDMQ") and other heteropolar polymers (see US 5,145,772), the AMPPD takes longer to reach the constant light emission characteristics than the optimal time periods ("t1/2", defined as the time required to reach half the maximum chemiluminescence intensity at constant, steady-state light emission levels; this emission half-time varies as a function of the stability of the dioxetane oxyanion in different environments).

Statistically, approximately seven t1/2 periods are required to reach the steady-state light emission kinetics.

The t1/2 for AMPPD at concentrations above 2 x 10-5 M in aqueous solution at pH 9.5 in the absence of BDMQ was found to be 7.5 minutes. The t1/2 at 4 x 10-3 M in the absence of BDMQ was found to be approximately 30-60 minutes, while the t1/2 for AMPPD at 2 x 10-5 M in aqueous solution was found to be 2.5 minutes.

In rapid bioassays using enzymatically cleavable chemiluminescent 1,2-dioxetanes as reporter molecules, it is desirable to reach steady-state light emission kinetics as quickly as possible to determine the "endpoint" in the assay. Chemiluminescence intensity can be measured before steady-state kinetics are reached. Sophisticated thermally controlled luminometry instruments must be used to obtain accurate values before steady-state emission kinetics.

AMPPD exhibits higher than desired thermal or other non-enzymatic activated light emission or "noise" in an aqueous buffered solution in both the presence and absence of chemiluminescent enhancers such as BDMQ. Such noise can be attributed to excited state emissions of adamantanone and the methyl-m-oxybenzoate anion derived from the aromatic portion of the AMPPD molecule. This noise can limit detection levels and thus prevent the realization of ultimate sensitivity since the measured noise content of AMPPD is approximately two orders of magnitude above the dark current in a standard luminometer.

Enzymatic cleavage of AMPPD with alkaline phosphatase also produces anionic dephosphorylated AMPPD - adamantylidene methoxymethylphenolate dioxetane or "AMP-D". This phenolate anion can also be formed hydrolytically in small amounts, producing a background chemiluminescent signal which causes greatly enhanced levels of light emission in an organized molecular arrangement such as in micelles, liposomes, lamellar phases, thin films, lipid bilayers, liposome vesicles, reverse micelles, microemulsion, microgel, latex, membrane or polymer surface and in hydrophobic environment such as that formed by chemiluminescent enhancers; e.g. BDMQ; thereby producing high background signals and substantially lowering the dynamic range of the signal formed by enzymatic hydrolysis of AMPPD.

In accordance with the observations described above, the applicant postulated the following mechanisms.

In the presence of reinforcing polymers such as BDMQ: 1. Enzymatic pathway (AP present): 2. Thermal route (no AP available):

1/ "CL" means chemiluminescence.

2/ AMPPD in aqueous buffered solution contains a small amount of dephosphorylated hydroxylated 1,2-dioxetane ("AMPHD"). If the solution pH is sufficiently high (above about 9.5), the dephosphorylated dioxetane may be present in anionic state, as AMPθD.

3/ "CL�1" and "CL₂" mean background chemiluminescence.

4/ "A*" means adamantantone in excited energy state.

Even in the absence of a reinforcing polymer, AMPPD can exist as an aggregate in aqueous solution: 3. Enzymatic pathway:

In the above mechanism, n>>>m; n and m are a function of the presence or absence of the reinforcing polymer and the AMPPD concentration.

The excited state of the adamantanone singlet in aggregate form (n or m > 1) can give higher yields of signal emission, again especially when "stabilized" to emit more light, as by the presence of a chemiluminescence enhancer such as BDMQ, than the excited energy state of the nonaggregated adamantanone. This is presumably due to the former exhibiting lower singlet states, lower yields of intersystem crossings, or slower intersystem crossings than the latter, or other factors not yet known. Since luminometers are generally designed to detect all photons, regardless of their energy or wavelength, 415 nm and 477 nm chemiluminescence are both detected as background noise emissions. Similarly, if photographic or X-ray film is used to record the When chemiluminescence is used, discrimination between the different wavelength emissions can be easily made, limiting the sensitivity of detection by background noise.

Finally, the observed aggregation of AMPPD under the conditions described above may be caused by the amphiphilic nature of AMPPD or its phenolate anion and similar molecules:

The present invention is therefore based on the object of reducing the time required to carry out assays and in particular bioassays in which enzymatically cleavable chemiluminescent 1,2-dioxetanes are used as reporter molecules. According to the invention, new and improved enzymatically cleavable chemiluminescent 1,2-dioxetanes are to be provided which, when used as reporter molecules in assays and in particular bioassays, reduce the time required to complete the assay.

It is an object of the invention to provide new and improved enzymatically cleavable chemiluminescent 1,2-dioxetanes that can be used as substrates for enzyme-based assays and in particular bioassays, which provide improved signal to background behavior and thus improved detection levels.

It is an object of the present invention to provide novel intermediates useful for the synthesis of these improved enzymatically cleavable 1,2-dioxetanes. It is an object of the present invention to provide processes for the preparation of these enzymatically cleavable chemiluminescent 1,2-dioxetanes and their intermediates. This and others are further explained and described in the following specification and in the appended claims.

Description of the invention

The invention relates to a new class of stable enzymatically cleavable chemiluminescent 3-(substituted adamant-2'-ylidene)-1,2-dioxetane compounds which can be cleaved in aqueous media, for example in a sample of a biological fluid in solution or on a solid surface, for example on a membrane surface such as a nylon membrane, with an enzyme or enzyme-modified specific binding pair and release optically detectable energy.

These modified adamantylidenedioxetanes enable assays in aqueous media using them as reporter molecules, with assays that can be performed more rapidly and with greater sensitivity than was possible in the past using AMPPD.

Although applicant does not wish to be bound by any mechanism or theory, this unexpected superior behavior can be explained by the fact that the presence of substituents of the type described here on or in the adamantylidene moiety prevents the dioxetane molecules from effectively packing and thus preventing them from forming "stabilized" organic composite forms, either in the form of micelles or in some other aggregated state. Certain of these substituents may be hydrogen bonded to other substances in their aqueous environment, including water itself, thereby further preventing aggregate formation. It is therefore possible that electronic and dipole effects contribute to this phenomenon, as indicated by the shorter t1/2 times and lower background noise.

Short description of the drawings

Figures 1-5 show TSH, RLU versus TSH for AMPPD and its bromo-, B-hydroxy-, A-hydroxy- and chloroadamant-2'-ylidene analogues obtained as described in Example XII below.

In Figures 6-10, the total luminescence emissions obtained from AMPPD and its A-hydroxy-, B-hydroxy-, chloro- and bromoadamant-2'-ylidene analogues obtained according to Example XIII are compared.

Figure 11 shows the improved chemiluminescence intensity obtained using chloroadamant-2'-ylidene analogue of AMPPD compared to AMPPD itself as a reporter molecule in a nucleic acid assay; see Example XIV below.

Figure 12 shows the kinetics of light emissions obtained using the chloroadamant-2'-ylidene analogue of AMPPD and AMPPD itself as reporter molecules in a nucleic acid assay; see Example XVI below.

Figure 13 is the dose response curve at five minutes for an alkaline phosphatase dilution with chloradamant-2'-ylidene analog of -AMPPD plus poly[vinyl(benzyldimethylammonium chloride)] ("BDMQ"), shown compared to the dose response curve at five minutes for AMPPD itself plus BDMQ; see Example XIX.

Figure 14 shows the dose response curves at 20 minutes for the same substances whose dose response curves are shown in Figure 13.

Fig. 15 is the dose response curve after 5 minutes for alkaline phosphatase dilution with the chloradamant-2'-ylidene analogue of AMPPD plus BDMQ-fluorescein ("Emerald") compared to the dose response curve at five minutes for AMPPD itself plus Emerald; see Example XIX below.

Fig. 16 shows the dose response curves at 20 minutes for the same substances whose dose response curves are shown in Fig. 15.

Figure 17 shows TPA sequence images obtained as described in Example XX below using (1) AMPPD and (2) AMPPD's chloroadamant-2'-ylidene analogue.

Figure 18 shows the signals obtained as in Example XII using (1) AMPPD and (2) substituted analogs of the invention.

Figure 19 shows the graphical representation of the chemiluminescence signal obtained according to Example XXVII obtained with (1) AMPPD and (2) Cl-AMPPD.

Figure 20 shows the exposure obtained according to Example XXVIX for the detection of pBR322 plasmid DNA using both (1) AMPPD and (2) Cl-AMPPD.

Figure 21 gives the exposures for the detection of pBR322 according to Example XXX using (1) AMPPD, (2) Cl-AMPPD and (3) Br-AMPPD.

Figure 22 is a graphical representation of the chemiluminescence signal obtained according to Example!!XXXI, plotted as a function of time for both (1) AMPPD and (2) Cl- AMPPD .

Figures 23 and 30 show the exposures obtained according to Examples!!XXXII and XXXIV in the detection of human transferrin for AMPPD, Cl-AMPPD and Br- AMPPD.

Figure 24 is a comparison in the signals obtained according to Example XXXV comparing AMPPD, Cl-AMPPD, Br-AMPPD and LumiPhos 530.

Figures 25 and 26 compare the images obtained according to Example XXXVI using AMPPD and Cl-AMPPD.

In Figures 27 and 28, the exposures obtained according to Examples XXXVIII and XXXIX are compared with the exposures obtained according to AMPPD, Cl-AMPPD and Br-AMPPD.

Fig. 29 shows the chemiluminescence detection of human transferrin according to Example 40 compared with AMPPD, Cl-AMPPD and Br-AMPPD.

Best mode for carrying out the invention

The new chemiluminescent 3-(substituted adamant-2'-ylidene)-1,2-dioxetanes according to the invention can be represented by the general formula:

being represented,

wherein X and X¹ each represent a substituent in the 5'- and 7'-positions of the adamant-2'-ylidene substituent, which individually represents a hydroxyl group, a halogen substituent, a -O(CH₂)nCH₃ group, wherein n = 0-19, a hydroxy(lower)alkyl group, a halo(lower)alkyl group, a phenyl group, a halophenyl group, an alkoxyphenyl group, a hydroxyalkoxy group, a cyano group, a carboxyl group or an amido group, wherein at least one of the substituents X and X¹ has a significance other than hydrogen; and

R₁ and R₂ individually or together represent an organic substituent which does not interfere with the generation of light when the dioxetane compound is enzymatically cleaved and which satisfies the valences of the 4-carbon atom of the dioxetane compound, with the provisos that when R₁ and R₂ represent individual substituents, the R₂ substituent is an aromatic, heteroaromatic, or an unsaturated substituent in conjugation with an aromatic ring, and that at least one of the substituents R₁ and R₂ or R₁ and R₂ together represent a fluorescent chromophoric group substituted with an enzymatically cleavable labile group which becomes luminescent when the enzymatically cleavable labile substituent is removed therefrom by an enzyme.

When the adamantylidene group is monosubstituted with any of the previously mentioned substituents, except hydrogen, a mixture of syn and anti isomers will be obtained when these compounds are synthesized. In certain cases, for example for monoiodo-substituted adamantylidenedioxetanes, one isomer will show a greater chemiluminescence intensity upon decomposition than the other. In other cases, for example for the monohydroxy-substituted adamantylidenedioxetanes, the two isomers will be equivalent or nearly so in their chemiluminescence properties, such as intensity. In any case, before being used, for example as reporter molecules in bioassays, the isomers can be separated by methods such as those described in US 5,342,966, or they can be used as chromatographed isomeric mixtures without separation.

Dioxetanes whose adamantylidene substituents are further substituted with two of the previously mentioned substituents excluding hydrogen (X plus X¹ ≠ hydrogen) do not show syn/anti isomerism, although of course positional isomers are possible when two different substituents are present, for example 5'-hydroxy-T-chloro- and 5'-chloro-7me-hydroxy-substituted adamantylidenedioxetanes.

The symbols R₁ and R₂ may represent any of the substituents on the 4-carbon atom of the dioxetane ring as described in WO 88/00695, US 5,089,630, WO 89/06226, US 4,952,707, US 5,145,772 and US 5,342,966 mentioned above, as long as R₁ and R₂ represent individual substituents, the R₂ substituent is an aromatic, heteroaromatic or unsaturated substituent in conjugation with the aromatic ring, and at least one of the substituents R₁ and R₂ or R₁ and R₂ together a fluorescence chromophore group is or are substituted with an enzymatically cleavable labile group which yields or yields a luminescent substance when the enzymatically removable labile substituent is removed therefrom by an enzyme.

For example, the symbol R1 may represent hydrogen or a bond when R2 represents a substituent which is bonded to the dioxetane ring via a spiro bond, or represents an organic substituent which does not interfere with the formation of light and which satisfies the valence of the dioxetane ring carbon atom to which it is bonded, giving a tetravalent dioxetane ring carbon atom, such as an alkyl, an aryl, aralkyl, heteroaryl, cycloalkyl or cycloheteroalkyl group, for example a straight-chain or branched-chain alkyl group having 1 to 7 carbon atoms inclusive; a straight-chain or branched-chain hydroxyalkyl group having 1 to 7 carbon atoms inclusive, an -OR- group wherein R represents a C1-C20 group. unbranched or branched unsubstituted or substituted, saturated or unsaturated alkyl, cycloalkyl, cycloalkenyl, aryl, aralkyl or aralkenyl group, each of which is additionally attached to R₂; may be condensed so that the emitting fragment contains a lactone ring, or an N-, O- or S-heteroatom-containing group, or an enzyme-cleavable group directly bonded to the 4-carbon atom of the dioxetane ring, or to one of the other R₁ groups mentioned above containing a bond cleavable by an enzyme, thereby forming, either directly or by subsequent adjustment of the pH, an electron-rich moiety, for example an oxygen anion, a sulfur anion or a nitrogen anion (the latter may be, for example, an oxime or an amido anion such as a sulfonamido anion) bonded to the dioxetane ring. Preferably R₁ represents an alkoxy group and in particular a methoxy group when R₂ is singly bonded to the 4-carbon atom of the dioxetane.

The symbol R₂ may also represent any organic substituent which does not interfere with the light generation and which satisfies the valence of the 4-carbon atom of the dioxetane ring to which it is attached. Preferably, R₂ will represent any number of light-emitting fluorophore-forming fluorescent chromophoric groups which enable the corresponding dioxetane decomposition fragments absorb energy and form an excited state from which they emit optically detectable energy and return to their ground state, substituted with an enzyme-cleavable group containing a bond cleavable by an enzyme, thereby forming, either directly or by subsequent adjustment of the pH, an electron-rich moiety, for example an oxygen anion, a sulfur anion or a nitrogen anion, bound to the dioxetane ring.

For example, the symbol R₂ alone (or together with the symbol R₁ a substituent spiro-bonded to the 4-carbon atom of the dioxetane ring) can represent fluorescent chromophoric groups such as:

- Phenyl and phenyl derivatives;

- naphthalene and naphthalene derivatives, for example 5-dimethylaminonaphthalene-1-sulfonic acid and hydroxynaphthalenes;

- anthracene and anthracene derivatives, for example 9, 10-diphenylanthracene, 9-methylanthracene, 9-anthracenecarboxaldehyde, anthryl alcohols and 9-phenylanthracene;

- Rhodamine and rhodamine derivatives, such as rhodole, tetramethylrhodamine, tetraethylrhodamine, diphenylmethylrhodamine, diphenyldiethyrhodamine and dinaphthylrhodamine

- fluorescein and fluorescein derivatives, for example 5-iodoacetamidofluorescein, 6-iodoacetamidofluorescein and fluorescein-5-maleimide;

- coumarin and coumarin derivatives, for example 7-dialkylamino-4-methylcoumarin, 4-bromomethyl-7-methoxycoumarin and 4-bromomethyl-7-hydroxycoumarin;

- Erythrosine and erythrosine derivatives, for example hydroxyerythrosine, erythrosyn-5-iodoacetamide and erythrosyn-5-maleimide;

- Acridine and acridine derivatives, such as hydroxyacridine and 9-methylacridine;

- Pyrene and pyrene derivatives, for example N-(1-pyrene)iodoacetamide, hydroxypyrene and 1-pyrenemethyliodoacetate;

- stilbene and stilbene derivatives, for example 6,6'-dibromostilbene and hydroxystilbenes;

- Nitrobenzoxadiazoles and nitrobenzoxadiazole derivatives, for example hydroxynitrobenzoxadiazoles, 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, 2-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)methylamino-acetaldehyde and 6-(7'-nitrobenz-2-oxa-1,3-diazol-4-yl)aminohexanoic acid;

- Quinoline and quinoline derivatives, for example 6-hydroxyquinoline and 6-aminoquinoline;

- acridine and acridine derivatives, for example N-methylacridine and N-phenylacridine;

- Acidoacridine and acidoacridine derivatives, for example 9-methylacidoacridine and hydroxy-9-methylacidoacridine;

- Carbazole and carbazole derivatives, for example N-methylcarbazole and hydroxy-N-methylcarbazole;

- fluorescent cyanines, for example DCM (a laser dye), hydroxycyanines, 1,6-diphenyl-1,3,5-hexatriene, 1-(4-dimethylaminophenyl)-6-phenylhexatriene and the corresponding 1,3-butadienes;

- carbocyanines and carbocyanine derivatives, for example phenylcarbocyanine and hydroxycarbocyanines;

- Pyridinium salts, for example 4-(4-dialkyldiaminostyryl)-N-methylpyridinium iodate and hydroxy-substituted pyridinium salts;

- Oxonols; and

Resorofine and hydroxyresorofine.

The symbol R₂ together with the symbol R₁ may also represent a condensed fluorescent chromophoric group bonded to the 4-carbon atom of the dioxetane ring via a spiro bond with the general formula:

the substituents R₄, R₅ and R₆ independently represent hydrogen, a branched or straight-chain alkyl group having 1 to 20 carbon atoms inclusive, for example methyl, n-butyl or decyl, a branched or straight-chain heteroalkyl group having 1 to 7 carbon atoms inclusive, for example methoxy, hydroxyethyl or hydroxypropyl; an aryl group having 1 or 2 rings, for example phenyl, naphthyl or anthryl; a heteroaryl group having 1 or 2 rings, for example pyrrolyl or pyrazolyl; a cycloalkyl group having 3 to 7 carbon atoms inclusive in the ring, for example cyclohexyl; a heterocycloalkyl group having 3 to 6 carbon atoms inclusive in the ring, for example dioxane; an aralkyl group having 1 or 2 rings, for example benzyl; or an alkaryl group having 1 or 2 rings, for example tolyl; and each R₃ independently represent hydrogen; an electron withdrawing group, such as a perfluoroalkyl group having 1 to 7 carbon atoms inclusive, for example trifluoromethyl; a halogen; CO₂H, -ZCO₂H, -SO₃H, ZSO₃H, NO₂, ZNO₂, -CN, or -Z₁CN, wherein Z is a branched or straight-chain alkyl group having 1 to 7 carbon atoms inclusive, for example methyl; an aryl group having 1 or 2 rings, for example phenyl; an electron donating group, for example a branched or straight-chain C₁-C₇ alkoxy group, for example methoxy or ethoxy; an aralkoxy group having 1 or 2 rings, for example phenoxy; a branched or straight-chain C₁-C₇ hydroxyalkyl group, for example hydroxymethyl or hydroxyethyl; a hydroxyaryl group having 1 or 2 rings, for example hydroxyphenyl; a branched or straight chain C₁-C₇ alkyl ester group, for example acetate; an aryl ester group having 1 or 2 rings, for example benzoate; or a heteroaryl group having 1 or 2 rings, for example benzoxazole, benzothiazole, benzimidazole or benzotriazole. Furthermore, two or more of the R₃ groups may form a fused ring or rings, which may themselves be unsubstituted or substituted.

The symbol R2, alone or together with the symbol R1, can also represent a special class of fluorophore moieties containing a fused polycyclic ring having a labile ring substituent containing a bond which, when cleaved, causes the fused polycyclic moiety to become electron rich and in turn renders the dioxetane compound decomposable to emit light. The members of this class are those in which the point of attachment of the labile ring substituent to the fused polycyclic ring, relative to that part or parts of the rings of the bond to the dioxetane ring (single bond, or when R1 represents a bond, a spiro bond) is so distinct that the total number of ring sp2 atoms separating these bond points, including the sp2 ring carbon atoms at the bond sites, is an odd integer; compare US 4 952 707.

Included among the fused polycyclic ring compounds whose residues can be used to form the fluorophore moiety are the fused polycyclic aromatic fluorophoric hydrocarbon ring compounds mentioned above, and particularly those containing from 9 to about 30 ring carbon atoms inclusive, such as naphthalene:

wherein the substituent bonds indicate a 1,6-substitution pattern as in disodium 6-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'-hydroxy)tricyclo[3.3.1.13,7]decan]-4-yl)-1- naphthalenyl phosphate, pentalene, azulene, heptalene, as-indacene; s-indacene, biphenylene, perylene, acenaphthylene, phenanthrene, anthracene, acephenanthrylene, aceanthrylene, triphenylene, pyrene, chrysol, naphthacene and the like, which also include the derivatives thereof substituted with one or more nonlabile substituents as mentioned above as examples for the symbols R₃, R₄ and R₅.

The fused polycyclic ring portion of such "odd pattern substituted" fluorophore moieties represented by R₂ alone or together with R₁ may also be the residue of nonaromatic" ie less fully aromatic fused polycyclic hydrocarbon ring fluorophore compounds having a labile ring substituent which containing a bond which, upon cleavage, renders the condensed less fully aromatic polycyclic moiety electron-rich, which in turn renders the dioxetane compound decomposable with light emission, unsubstituted or substituted with one or more of the aforementioned non-labile substituents, and containing 10 to about 30 ring carbon atoms inclusive, such as fluorene, 3,4-dihydro-3,3-dimethylnaphthalene, dibenzosuberene, 9,10-dihydrophenanthrene, indene, indeno[1,2-a]indene, phenalene, fluoranthrene and the like.

The fused polycyclic ring portion of the fluorophore moieties represented by R₂ alone or together with R₁, may also be the residue of a fused polycyclic heteroaromatic or less than fully aromatic fused ring heterocyclic fluorophore forming group, for example dibenzothiophene, dibenzofuran, 2,2-dimethyl-2Hchromene, xanthene, piperidine, quinoline, isoquinoline, phenanthridine, carbostyryl, phenoxazine, phenothiazine, phenanthroline, purine, phthalazine, naphthyridine, N-acylindole, chromane, isochroman, N-acylindoline, isoindoline and the like, unsubstituted or substituted with one or more of the aforementioned non-labile substituents and containing from 9 to about 30 ring atoms inclusive, the majority of which are carbon atoms.

A preferred enzymatically removable group with which at least one of R₁ and R₂ is substituted is a phosphate group, particularly a phosphate ester group, represented by the general formula:

wherein M+ is a cation such as an alkali metal, for example sodium or potassium, ammonium or a C₁-C₇ alkyl, aralkyl or aromatic quaternary ammonium cation, N(R₇)₁₄, where R₇ is alkyl, for example methyl or ethyl, aralkyl, for example benzyl, or part of a heterocyclic ring system, for example pyridinium. The disodium salt is particularly preferred. Such phosphate ester groups can be cleaved using an enzyme such as alkaline phosphatase to form oxygen anion substituted groups which in turn destabilise the dioxetane with cleavage of its oxygen-oxygen bond under photoemission. The quaternary ammonium cations in such phosphate groups can also have one of their quaternary groups attached to a polymeric backbone such as

where n is greater than 1, or they may form part of a polyquaternary ammonium salt, for example an ionene polymer.

Another preferred enzymatically removable group is the β-D-galactoside group, which can be cleaved with the enzyme β-D-galactosidase to form the conjugate acid of dioxetane phenolate, which exhibits chemiluminescence under pressure.

Enzymatically cleavable substituents that may also be used include enzyme-cleavable alkanoyloxy groups, for example an acetate ester group, or an enzyme-cleavable oxacarboxylate group, 1-phospho-2,3-diacylglyceride group, 1-thio-D-glucoside group, adenosine triphosphate analogue group, adenosine diphosphate analogue group, adenosine monophosphate analogue group, adenosine analogue group, α-D-galactoside group, β-D-galactoside group, α-D-glucoside group, β-D-glucoside group, α-D-mannoside group, β-D-mannoside group, β-D-fructofuranoside group, β-D-glucosiduronate group, p-toluenesulfonyl-larginine ester group or p-Toluenesulfonyl-L = arginine amide group.

When R₂ is phenyl, as indicated, R₁ may be -O(CH₂)nCH₃ when n is 0-19, preferably 0-5. In such compounds, important properties can be further imparted or controlled in the molecule by additional substitution on the phenyl ring. The stability, solubility, aggregation, binding ability and decomposition kinetics can be further controlled in compounds of the following structure

wherein Z is the enzyme-cleavable group discussed above, the oxygen is in ortho, meta or para, and Q is hydrogen, aryl, substituted aryl, aralkyl, heteroaryl with up to 20 carbon atoms, an allyl group, a hydroxy(lower)alkyl, a lower-alkyl-OSiR³ (wherein R₃ is medium-alkyl, aryl, alkoxy-C₁₋₆, alkoxyalkyl with 12 or less ger carbon atoms, hydroxy(lower)alkyl, amino(lower)alkyl, -OR⁴ or -SR⁴ (wherein R₄ is alkenyl, substituted (lower)alkenyl, (lower)alkyl or aralkyl having up to 20 carbon atoms, -SO₂R⁵ (wherein R�5 is methyl, phenyl or NHC₆H₅), substituted or unsubstituted lower alkyl, nitro, cyano, halogen, hydroxy, carboxyl, trimethylsilyl or a phosphoryloxy group.

PCT Application No. WO88/00695, published on January 28, 1988, describes enzymatically cleavable 1,2-dioxetanes substituted at the 3-carbon atom with a defined substituent "T-V" and at the 4-carbon atom with defined substituents "X" and "Y-Z". In this published application, on page 3, lines 6-12, it is described:

According to preferred embodiments, one or more groups T, X or Y further comprise a solubilizing substituent, for example a carboxylic acid, a sulfonic acid, a quaternary amino salt; the group T of the dioxetane is a polycycloalkyl group, preferably adamantyl; the enzyme-cleavable group comprises phosphate; and the enzyme has phosphatase activity.

on page 22, line 33 to page 23 line 6 it is described:

For example, the enzyme-cleavable group Z can be bound to the group X of the dioxetane instead of the group Y. The specific affinity substance can be bound to the dioxetane via groups X, Y or T

(preferably the group X) instead of the enzyme. In this case, the group to which the specific affinity substance is bound includes, for example, a carboxylic acid, amino or maleimide substituent to facilitate binding.

and on page 23 lines 11-21 described:

The groups X, Y or T of the dioxetane can be bonded to a polymerizable group, for example a vinyl group, which can be polymerized to form a homopolymer or copolymer.

The X, Y or T groups of the dioxetane can be attached to, for example, membranes, films, beads or polymers so that they can be used in immuno- or nucleic acid assays. The groups contain, for example, carboxylic acid, amino or maleimide substituents to facilitate attachment.

The X, Y or T groups of the dioxetane can contain substituents that enhance the kinetics of the enzymatic dioxetane degradation, for example electron-rich groups (e.g. methoxy).

The groups Y and T of the dioxetane, as well as the group X, can contain solubilizing substituents.

The problem solved by the 3-(substituted adamant-2'-ylidene)-1,2-dioxetane compounds described and claimed herein is not addressed in this published PCT application, nor are these compounds themselves disclosed in this or any other reference known to the inventors.

The overall synthesis of these 3-(substituted adamant-2'-ylidene)-1,2-dioxetanes can be carried out using the methods described in WO 88/00695, WO 89/06226 and WO 91/03479.

For example, 1,2-dioxetanes falling within the scope of Formula I above, wherein X is a hydroxyl group, X¹ is a hydrogen, R₁ is a methoxy group and R₂ is a phosphoryloxy salt substituted phenyl group, preferably a meta-phosphoryloxy salt substituted phenyl group, can be synthesized according to the methods described in the '176 application by a reaction sequence which can be schematically represented as follows:

As specifically described below by way of example, the O = φOR�9 starting material may, if desired, be reacted with an orthoformate such as trimethyl orthoformate, methanol, and p-toluenesulfonic acid to give the intermediate:

which is then reacted with (R�8O)₃P and a Lewis acid to give the phosphonate ester intermediate:

is received.

In the reaction sequence mentioned above, R8 represents a lower alkyl group, for example methyl, ethyl or butyl. R9 represents an acyl group containing 2 to about 14 carbon atoms inclusive, such as acetyl, propionyl, mesitoyl or pivaloyl, Q represents a halogen, for example chlorine or bromine or ORg, and M independently represents a proton, a metal cation, for example Na+ or K+, or an ammonium, substituted ammonium, quaternary ammonium or (H+)-pyridinium cation. Thiolate cleavage as described in WO89/06226 can be used instead of base cleavage of the OR9 group in step 5 of the reaction sequence explained above, in which case R9 represents represents a lower alkyl, lower alkenyl or aralkyl group, for example methyl, allyl or benzyl. The product of the base or thiolate cleavage may contain, instead of R�9, a hydrogen or an alkali metal cation, for example lithium, sodium or potassium.

The intermediates described above by the formula

where only one of X and X¹ is other than hydrogen, are known compounds, or they can be readily synthesized from known starting materials using known methods. For example, in the case of the monosubstituted adamantane-2-ones (one of X and X¹ is hydrogen):

- 5-Hydroxyadamantan-2-one, prepared as described in Geluk, Synthesis 374 (1972) ;

- 5-Bromoadamantan-2-one and 5-chloroadamantan-2-one, prepared as described in Geluk et al., Tetrahedron 24 5369 (1968).

When X or X¹ is fluorine, unsubstituted (lower)alkyl, for example t-butyl, substituted (lower)alkyl, for example trifluoromethyl, unsubstituted aryl, for example phenyl or substituted aryl, for example p-chlorophenyl, p-methoxyphenyl or p-nitrophenyl, see le Noble et al., J. Am. Chem. Soc. 108 1598 (1986) and Walborsky, et al., J. Am. Chem. Soc., 109 6719 (1987) (X or X¹ = hydroxymethyl).

Simple unit procedures allow the conversion of various of the aforementioned X or X¹ substituents or other known ones into 5-X- or 5-X¹-adamantan-2-ones, where X or X¹ represents trialkylsilyloxy, iodo or cyano groups: These moieties are stable under the mild conditions used in step 4 of the aforementioned reaction sequence. For example, when 5-hydroxyadamantan-2-one is refluxed with 57% hydroiodic acid for 7 hours, 5-iodoadamantan-2-one (m.p. 73-76°C) is obtained. 5- Carboxyadamantan-2-one prepared as described in Lantvoev, J. Obshch. Khim. 2 2361 (1976) or Le Noble et al., J. Org. Chem., 48 1101 (1983), after saponification of the methyl ester, it can be converted to 5-cyanoadamantan-2-one according to a three-step procedure of Tabushi et al., J. Org. Chem., 38 3447 (1973), which is used to access the isomeric 1-cyanoadamantan-2-one via a ketoamide intermediate. 5- Trimethylsilyloxyadamantan-2-one (m.p. 34-38 °C), which is useful as a protected version of 5-hydroxyadamantan-2-one, allows the use of only one equivalent of base in step 4 of the preceding reaction sequence to produce the corresponding enol ether, which can then be desilylated using standard procedures.

As will be appreciated by those skilled in the art, other X and X¹ groups need not be static throughout the reaction sequence, but may be transformed by reactions compatible with other structural considerations at any time. For example, it has been found that when X or X¹ represents a chlorine or a bromine atom, the enol ether intermediates formed in steps 4 and 5 of the aforementioned reaction sequence readily undergo solvolysis in the presence of molecular excesses of diols or liquid ammonia in a bomb at high temperature. The reaction rate with diols such as ethylene glycol or propylene glycol only becomes pronounced at elevated temperatures (105-120°C) in the presence of a proton acceptor such as potassium carbonate. In general, this reaction is slow but clean, and the use of silver or Heavy metal salts are avoided, which are often used to stimulate hydroxyalkyl ether formation. 3-(Methoxy-5-(2-hydroxyethoxy)tricyclo[3.3.1.13,7]dec-2-ylidenemethyl)phenol can be esterified with trimethylacetyl chloride and triethylamine to give the corresponding diester, which can then be selectively cleaved using potassium carbonate in methanol to give the phenol monoester. The corresponding phosphorylation step, which involves simultaneous β elimination and saponification of the hindered ester with sodium methoxide in methanol, gives the hydroxyethoxy enol ether phosphate.

Reaction with liquid ammonia in dioxane under pressure gives 3-(methoxy-5- aminotricyclo[3.3.1.13,7]dec-2-ylidenemethyl)phenol from the corresponding 5-bromo compound and is carried out using the procedure described by Hummelen, Dissertatiozt University of Groningen, Netherlands, p. 60 (1985). The immediate acylation of the amino enol ether phenol thus obtained using two equivalents of acetyl chloride or acetic-formic anhydride and 4-dimethylaminopyridine as base according to the procedure of Gawronski; et al., J. Am. Chem. Soc., 109 6726 (1987), used for the esterification of (5-hydroxyadamantylidene)ethanol, gives the formamido or acetamidophenol esters, which can be selectively saponified as described above and then phosphorylated and photooxygenated as described below.

Meijer, Dissertation University of Groningen, Netherlands (1982); Numan et al., J. Ora. Chem., 43 2232 (1978); and Faulkner, et al., J. Chem. Soc. Chem. Comm., 3906 (1971) describe the access to 4-methyleneadamantan-2-one (X and X¹ hydrogen, methylene in the 4'- position of formula I above) as starting material for step 4 of the aforementioned reaction sequence and subsequent reaction with a suitable phosphonate-stabilized carboxy ion. The difference in the reactivity of the singlet oxygen towards the enol ether instead of the exomethylene function ensures that disodium 3-(4-methoxyspiro-[1,3-dioxetane- 3,2'-(4'-methylene)tricyclo[3.3.1.13,7]decan]-4-yl)phenyl phosphate is obtained as the photooxygenation product.

When a selectively cleavable pivaloyloxyaryl enol ether is obtained in any of the reactions immediately preceding the addition of the enzyme-removable group, such as the phosphate ester group, it is more convenient to avoid isolation of the hydroxyaryl enol ether. This can be accomplished by directly cleaving the pivaloyl ester with one equivalent of sodium methoxide in methanol and isolating the sodium aryloxide species as a dry solid by removing all volatiles upon completion of the reaction. In such a case, Step 6 of the reaction sequence described above is carried out using this preformed salt in a dry, polar aprotic solvent such as dimethylformamide without using a Lewis base, and the inorganic salt byproducts are removed in the work-up phase of Step 7 or Step 8.

The 2-cyanoethyl phosphate diester product of step 7 undergoes β elimination to the phosphate monoester of step 8. In step 8, derivatives where X or X¹ = chlorine or bromine, preferably with a volatile amine such as ammonia or with a solvent soluble organic amine such as "DBU" (1,8 diazabicyclo[5.4.0]undec-7-ene) in an alcohol solvent, for example methanol. The use of ammonia at atmospheric pressure or above and at ambient temperatures is particularly advantageous since the excess base is simply evaporated in vacuo at the end of the reaction.

The oxidation of the enol ether phosphate at step 9 of the previously described reaction sequence can be carried out photochemically as indicated by reaction with singlet oxygen (102) in a halogenated solvent, for example a halogenated hydrocarbon, such as chloroform, which may also contain a co-solvent, for example a lower alkanol such as methanol. Singlet oxygen can be generated using a photosensitizer such as polymer-bound Rose Bengal (Polysciences, Inc.), methylene blue or 5-,10-,15-,20-tetraphenyl-21H,23H-porphine ("TPP").

Alternatively, the crude 2-cyanoethyl phosphate diesters obtained in step 7 of the aforementioned reaction sequence can be oxidized with singlet oxygen to their 1,2-dioxethane counterparts. Subsequent reaction with sodium methoxide in methanol at room temperature allows aqueous work-up and preparative reversed-phase high-pressure liquid chromatography to yield pure 1,2-dioxetane phosphate monoester salts as mixtures of their syn and anti isomers. Chemical methods for 1,2-dioxetane formation include those using triethylsilyl hydrotrioxide, phosphatozonides, or one-electron oxidation of triarylamine radical cations mediated in the presence of triplet oxygen can also be used.

Starting materials of the formula:

wherein X and X¹ are both the same and have a meaning other than hydrogen, or intermediates from which such starting materials can be synthesized using methods known per se are also known. For example, the use of symmetrically substituted 5,7-bis-X,X'-adamantan-2-ones in step 4 of the aforementioned reaction sequence gives symmetrical 1,2-dioxetanes, such that X and X' have substituents in both syn and anti relationships to the four-membered dioxetane ring. The quinone monoacetate-based synthetic strategy of Stetter et al. gives access to 5,7-dihydroxyadamantan-2-one via bicyclo[3.3.1]nonane-3,7-dione-9-ethylene acetal; Steifer, et al., Liebigs Ann. Chem., 1807 (1977); see also Hamill, et al., Tetrahedron, 27 4317 (1971). 5,7-Dihydroxyadamantan-2-one can be converted to 5,7-dibromoadamantan-2-one or its 5,7-dichloro and 5,7-diiodine analogues using 47% aqueous hydrobromic acid, thionyl chloride or 57% aqueous hydriodic acid under the conditions described by Geluk, et al., supra. 5,7-Dialkyladamantan-2-ones, e.g. 5,7-dimethyladamantan-2-one, can be synthesized according to the method of Kira, et al., J. Am. Chem. Soc., 111 8256 (1989). Solvolysis of 3-(methoxy-5,7-dibromotricyclo[3.3.3.13,7]dec-2-ylidenedimethyl)phenol with diols such as ethylene glycol or 1,4-butanediol in the presence of potassium carbonate also gives the corresponding symmetrical bishydroxyalkoxy-substituted 1,2-dioxetanes using a modified route to the monosubstituted derivatives as described above.

When one wishes to take advantage of the joint effects of an X group other than hydrogen which is different from the X¹ group which is also present and which is also other than hydrogen, particularly when advantages can be obtained using such enzymatically cleavable 1,2-dioxetanes as isomeric mixtures, unsymmetrical 5,7(X,X¹)adamantan-2-ones are used in step 4 of the preceding reaction sequence. 5-Bromo-7-trifluoradamantan-2-one can be prepared according to the procedure described by Sorochinskii, et al., Zh. Obshch. Khim., 7 2339 (1981). 5-Chloro-7-hydroxyadamantan-2-one and 5-methyl-7-hydroxyadamantan-2-one were described by Stetter, et al., supra, and 5-bromo-7-hydroxyadamantan-2-one can be synthesized from 7-methylenebicyclo[3.3.1]nonane-3,9-dione-9-ethyleneacetal according to the method of Stetter, et al., by dissolving this compound in anhydrous ethanol and saturating the solution at 0°C with gaseous hydrogen bromide instead of gaseous hydrogen chloride used to prepare the corresponding chloro derivative.

Intermediates and processes for synthesizing compounds of formula I above, wherein R₁ is other than lower alkoxy and R₂ is other than phosphoryloxy salt substituted phenyl, are described in the aforementioned WO 88/00695, U.S. 5,089,630, WO 89/06226 and U.S. 4,952,707.

The present invention, as indicated above, also relates to the use of chemiluminescent, enzymatically cleavable substituted 1,2-dioxetanes in known assays, including assays for detecting enzymes in samples. The invention further relates to kits for use in such assays and similar uses and means for carrying out such uses.

For example, when the present invention is used to detect an enzyme in a sample, the sample is treated with a dioxetane bearing a group that is cleavable by the enzyme to be detected. The enzyme cleaves the enzyme-cleavable group of the dioxetane to form a negatively charged substituent (e.g., an oxygen anion) bound to the dioxetane. This negatively charged substituent in turn destabilizes the dioxetane and causes the dioxetane to decompose to form a fluorescent chromophoric group that emits light energy. It is this chromophoric group that is detected as an indication of the presence of the enzyme. By measuring The intensity of the luminescence can also determine the concentration of the enzyme in the sample.

A large number of other assays exist which use visually detectable means to determine the presence or concentration of a particular substance in a sample. The dioxetanes described above can be used in any of these assays. Examples of such assays include immunoassays for detecting antibodies or antigens, for example α- or β-hCG, enzyme assays, chemical assays for detecting, for example, potassium or sodium ions, and nucleic acid assays for detecting, for example, viruses (for example, HTLV III or cytomegalovirus or bacteria (for example, E. coli) and certain cell functions (for example, receptor binding sites).

When the detectable substance is an antibody, an antigen or a nucleic acid, the enzyme capable of cleaving the enzyme-cleavable group of the dioxetane is preferably bound to a substance having a specific affinity for the detectable substance (i.e. a substance that specifically binds to the detectable substance), for example an antigen, an antibody or a nucleic acid probe. Known methods, for example carbodiimide coupling, are used to bind the enzyme to the specific affinity substance, where the binding preferably occurs via an amide linkage.

In general, the assays are performed as follows. A sample suspected of containing a detectable substance is contacted with a buffered solution containing an enzyme bound to a substance having a specific affinity for the detectable substance. The resulting solution is incubated to allow the detectable substance to bind to the specific affinity portion of the specific affinity enzyme compound. Excess specific affinity enzyme compound is then washed away and a dioxetane having a group cleavable by the enzyme portion of the specific affinity enzyme compound is added. The enzyme cleaves the enzyme cleavable group and causes the dioxetane to decompose into two carbonyl compounds, such as an ester, a ketone or an aldehyde. The chromophore to which the enzyme-cleavable group was bound is thus excited and luminesces. The luminescence is detected (using, for example, a cuvette or a light-sensitive film in a camera luminometer, or a photoelectric cell or photomultiplier tube as an indication of the presence of the detectable substance in the sample. The luminescence intensity is measured to determine the concentration of the substance.

According to the invention there is provided an assay method which uses an optically detectable reaction to determine the presence or absence of a particular substance in the sample, wherein the optically detectable reaction comprises the reaction with an enzyme of an enzymatically cleavable chemiluminescent 1, 2 = dioxetane compound represented by the formula

where the substituents are as defined above.

According to a preferred embodiment, the substituents X and X¹ in the formula are chlorine and hydrogen, respectively. Disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2'- (5'-chloro)tricyclo[3.3.1.13,7]decan]-4-yl)phenyl phosphate is preferably used.

As the aforementioned specific binding pairs, the following are preferably used in the assay method of the invention:

(i) an antigen and an antibody,

(ii) a nucleic acid and a probe capable of binding part or all of the nucleic acid,

(iii) an enzyme and a 1,2-dioxetane compound containing a group cleavable by the enzyme.

Preferably, the aforementioned probe is a labeled oligonucleotide complementary to the nucleic acid. DNA, RNA and fragments thereof can be generated according to a sequencing protocol.

This preferred assay method further comprises the steps of (a) treating the DNA, RNA or a fragment thereof with a labeled complementary oligonucleotide probe to form a hybridization pair, (b) treating the hybridized pair with a molecule strongly bound to the label of the oligonucleotide that is covalently conjugated to an enzyme capable of cleaving an enzymatically cleavable 1,2-dioxetane with the release of light energy,

(c) addition of a 1,2-dioxetane substrate and

(d) Detection of the light produced.

Preferably, the oligonucleotide label is biotin or a biotin derivative. The molecule that can interact strongly with the oligonucleotide label is preferably avidin or streptavidin.

The previously mentioned covalent labeling for the oligonucleotide probe is desirable, the use of an enzyme that can decompose the 1,2-dioxetane compound with light energy emission.

According to a preferred embodiment of the invention, the assay method is a method in which the label on the oligonucleotide probe comprises a covalently bound antigen immunochemically linked to an antibody-enzyme conjugate, the antibody being directed against the enzyme and the enzyme being capable of decomposing the 1,2-dioxetane compound with the emission of light energy.

According to a preferred embodiment, the enzyme is an acid or alkaline phosphatase, R₁ is methoxy and R₂ is a metaphosphate-substituted phenoxy group

The probe is preferably bound to the nucleic acid using a nylon membrane. The same applies to the hybridization between DNA, RNA or a fragment thereof and the labeled oligonucleotide probe.

In a preferred embodiment of the assay method according to the invention, a solid matrix is used in which non-specific binding to the matrix is blocked by pretreatment of the matrix with a polymeric quaternary ammonium salt. It is further desirable to further carry out the assay method in the presence of a water-soluble enhancing substance which enhances the specific light energy production above the value in its absence. As an example of such an enhancing substance, a polymeric quaternary ammonium salt can be mentioned. Preferred polymeric quaternary ammonium salts are selected from poly(vinylbenzyltrimethylammonium chloride), poly[vinylbenzyl(benzyldimethylammonium chloride)] or poly[vinyl(benzyltributylammonium chloride)].

A preferred method is a method in which the enhancing substance comprises a positively charged polymeric quaternary ammonium salt and fluorescein which can form a ternary complex with the negatively charged product of the 1,2-dioxetane compound formed after the enzyme-catalyzed decomposition of the 1,2-dioxetane compound, whereby energy transfer occurs between the negatively charged product and fluorescein and whereby light is emitted from fluorescein. The use of the aforementioned quaternary ammonium salts is also preferred in this method.

Examples of specific assays follow. A. Assay for human IgG

A 96-well microtiter plate is coated with a sheep anti-human IgG (F(ab)2 fragment specific). A serum sample containing human IgG is then added to the wells and the wells are incubated for 1 hour at room temperature.

After the incubation period, the serum sample is removed from the wells and the wells are washed four times with an aqueous buffer solution containing 0.15 M NaCl, 0.01 M phosphate and 0.1% bovine serum albumin (pH 7.4).

Alkaline phosphatase conjugated to anti-human IgG is added to each well and the wells are incubated for 1 hour. The wells are then washed four times with the above buffer solution and a buffer solution of phosphate-containing dioxetane according to the invention is added. The resulting luminescence, which is caused by enzymatic degradation of the dioxetane, is detected with a luminometer or with a photographic film in a camera luminometer.

B. Assay for hCG

Rabbit anti-α-hCG is adsorbed to a nylon mesh membrane. A sample solution containing hCG, for example, urine from a pregnant woman, is blotted through the membrane, after which the membrane is washed with 1 ml of buffer solution containing 0.15 M NaCl, 0.01 M phosphate and 0.1% bovine serum albumin (pH 7.4).

The alkaline phosphatase-labeled β-hCG is added to the membrane and the membrane is washed again with 2 ml of the above buffer solution. The membrane is then placed in the cuvette of a luminometer or in a camera luminometer and contacted with a phosphate-containing dioxetane according to the invention. The luminescence that occurs due to the enzymatic degradation of the dioxetane is then detected.

C. Serum alkaline phosphatase assay

2.7 ml of an aqueous buffer solution containing 0.8 M 2-methyl-2-aminopropanol is placed in a 12 x 75 mm Pyrex test tube and 0.1 ml of serum sample containing alkaline phosphatase is added. The solution is then equilibrated at 30°C. 0.2 ml of a phosphate-containing dioxetane according to the invention is added and the test tube is immediately placed in a luminometer to record the resulting luminescence. The amount of light emission is proportional to the rate of alkaline phosphatase activity.

D. Nucleic acid hybridization assay

A sample of cerebrospinal fluid (CSF) suspected of containing cytomegalovirus is collected and placed on a membrane, such as a nylon or nitrocellulose membrane. The sample is then chemically treated with urea or guanidinium isothiocyanate to break down the cell walls and degrade all cellular components except viral DNA. The strands of viral DNA thus formed are separated and bound to the nitrocellulose filter. A DNA probe specific for the viral DNA and labeled with alkaline phosphatase is then applied to the filter; the probe hybridizes with the complementary viral DNA strands. After hybridization, the filter is washed with an aqueous buffer solution containing 0.2 M NaCl and 0.1 mM Tris-HCl (pH = 8.10) to remove excess probe molecules. A phosphate-containing dioxetane according to the invention is added and the resulting luminescence from the enzymatic degradation of the dioxetane is measured in a luminometer or detected with a photographic film.

E. Assay for Galactosidase

In the assays described above and in the working examples below, dioxetanes containing α- or β-galactosidase-cleavable α-D- or β-D-galactoside (galactopyranoside) groups can be added and the luminescence produced by the enzymatic cleavage of the sugar moiety from the chromophore is measured in a luminometer or detected with photographic film.

F. Electrophoresis

Electrophoresis allows the separation of complex mixtures of proteins and nucleic acids according to their molecular sizes and structures on gel supports in an electric field. This technique can also be used to separate protein fragments after proteolysis or fragments of nucleic acids after cleavage by restriction endonucleases (as in DNA sequencing). After electrophoretic separation of the species in a gel or after transfer of the separated species from a gel to a membrane, the samples are treated with a probe of an enzyme bound to a ligand. For example, peptide fragments are probed with an antibody covalently bound to alkaline phosphatase. As another example, in DNA sequencing, alkaline phosphatase-avidin binds to biotinylated nucleotide bases. An AMPPD analogue of the invention is then added to the gel or membrane filter.

After a short incubation, light is emitted as a result of enzymatic activation of the dioxetane to form the corresponding emitting species. The luminescence is detected either by X-rays or by instant photographic film or by scanning with a luminometer. Multi-channel analysis further improves the method, making it possible to probe more than one fragment at a time.

G. Solid State Assays

In solid state assays, it is desirable to block nonspecific binding to the matrix by pretreating the nonspecific binding sites with nonspecific proteins such as bovine serum albumin (BSA) or gelatin. Some commercially available preparations of BSA have been found to contain small amounts of substances having phosphatase activity, thereby producing undesirable background chemiluminescence from AMPP. Certain water-soluble synthetic macromolecular substances have also been found to be efficient blocking agents for nonspecific binding in solid state assays using dioxetanes. Preferred among such substances are water-soluble polymeric quaternary ammonium salts such as BDMQ, poly(vinylbenzyltrimethylammonium chloride) (TMQ) and poly(vinylbenzyltributylammonium chloride) (TBQ). Other such substances are described in the aforementioned Voyta, et al. '263 application and are listed in Table III below.

H. Assay for nucleotidase

An assay for the enzyme ATPase is performed in two steps. In the first step, the enzyme is treated at optimal pH (typically pH 7.4) with a substrate containing ATP, covalently bound via a terminal phosphoester bond to a chromophore-substituted 1,2-dioxetane to produce phosphoryl-chromophore-substituted 1,2-dioxetane. In the second step, the product of the first step is decomposed by adding an acid to adjust the pH below 6, preferably to pH 2 to 4, and the resulting light is measured in a luminometer or detected with a chromatographic film. In a similar two-step process, ADPase is analyzed using as a substrate an ADP derivative of a chromophore-substituted 1,2-dioxetane of the invention, and 5'-nucleotidase is analyzed using an adenylic acid derivative as a substrate of a chromophore-substituted 1,2-dioxetane of the invention. The second step can also be carried out by adding the alkaline enzyme phosphatase to decompose the phosphoryl-chromophore-substituted 1,2-dioxetane.

I. Nucleic acid sequencing

DNA or RNA fragments formed in sequence protocols can be detected after electrophoretic separation using chemiluminescent 1,2-dioxetanes according to the invention.

DNA sequencing can be performed according to a dideoxy chain termination procedure [Sanger, F., et al., Proc. Nat. Acad. Sci. (USA), 74: 5463 (1977)]. Briefly, for each of the four sequencing reactions, single-stranded template DNA is mixed with deoxynucleotides and biotinylated primer strand DNA. After annealing, Klenow enzyme and deoxyadenosine triphosphate are incubated with each of the four sequencing reaction mixtures, and then chase deoxynucleotide triphosphate is added and incubation is continued.

The DNA fragments in the reaction mixtures are then separated using polyacrylamide gel electrophoresis (PAGE). The fragments are then transferred to a membrane, preferably a nylon membrane, and the fragments are bound to the membrane by exposure to UV light, preferably with a short wavelength.

After blocking the non-specific binding sites with a polymer, e.g. heparin, casein or serum albumin, the DNA fragments on the membrane are contacted with avidin or streptavidin covalently linked to an enzyme specific for the enzyme-cleavable group of the particular 1,2-dioxetane substrate of the invention being used. Since avidin or streptavidin binds strongly to biotin, biotinylated DNA fragments are now attached to an enzyme. For example, if the chemiluminescent substrate is disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan]-4-yl)phenylphosphate dioxetane salt (Cl-AMPPD), avidin or streptavidin is conjugated to a phosphatase. Similarly, if the chemiluminescent substrate is disodium 3-(4-methoxyspiro[1,2- dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan]-4-yl)phenyl-β-D-galactopyranose (Cl-AMPGD), avidin or streptavidin is conjugated to galactosidase.

After generating chemiluminescence by treating the DNA fragment-biotin-avidin (or streptavidin) enzyme complex with the appropriate 1,2-dioxetane at alkaline pH values, e.g., above about 8.5, the DNA fragments can be visualized with a light-sensitive film, e.g., X-ray or instant film, or in a photoelectric luminometer instrument.

The detection method described above can also be applied to the genomic DNA sequence protocol of Church, et al., [Church, G. M., et al., Proc. Nat. Acad. Sci. (USA), 81: 1991 (1984)]. After transferring the chemically cleaved and electrophoretically separated DNA [Maxam, A. M. et al., Proc. Nat. Acad. Sci. (USA), 74: 560 (1977)] to a membrane, preferably a nylon membrane, and cross-linking the ladders to the membrane using UV light, specific DNA sequences can be detected by sequential addition of: biotinylated oligonucleotides as hybridization probes; Avidin or streptavidin, covalently linked to an enzyme specific for the enzyme-cleavable chemiluminescent 1,2-dioxetane of the invention and the appropriate 1,2-dioxetane: Images of the sequence ladders (generated by PAGE) can be obtained as described above. Serial re-probe formation of sequence ladders can be performed by first stripping the hybridized probe and chemiluminescent material from the membrane by treating the membrane with a heated solution of detergent, e.g., from about 0.5 to about 5% sodium dodecyl sulfate (SDS) in water at about 80°C to about 90°C, cooling from about 50°C to about 70°C, hybridizing the now naked DNA fragments with another biotinylated oligonucleotide probe to produce a different sequence, and then generating imaging chemiluminescence as described above.

Similar detection methods can be performed with RNA fragments generated by RNA sequencing techniques.

The invention further relates to a kit for the detection of a first substance in a sample, comprising an enzymatically cleavable chemiluminescent 1,2-dioxetane compound which can generate light energy upon decomposition, represented by the formula

wherein the substituents R₁, R₂, X and X¹ have the meanings given above. In this kit, a 1,2-dioxetane compound is preferably used, wherein R₁ is methoxy, R₂ is a meta-phosphate-substituted phenoxy group and the enzyme is acid or alkaline phosphatase.

The invention further relates to a kit for detecting a nucleic acid or a fragment thereof in a sample by hybridizing the nucleic acid or fragment to a complementarily labeled oligonucleotide probe comprising an enzymatically cleavable chemiluminescent 1,2-dioxetane compound as defined above, an oligonucleotide probe covalently labeled with an enzyme and a nylon membrane on which the nucleic acid-oligonucleotide probe hybridization is carried out. Such a kit is preferred wherein R₁ is methoxy, R₂ is a meta-phosphate-substituted phenoxy group and the enzyme is an acid or alkaline phosphatase.

The invention further relates to a kit for detecting a nucleic acid or a fragment thereof in a sample by hybridizing the nucleic acid or a fragment thereof with a complementarily labeled oligonucleotide probe. The kit comprises an enzymatically cleavable chemiluminescent 1,2-dioxetane compound as defined above, a complementary oligonucleotide probe covalently labeled with a biotin or biotin derivative, avidin or streptavidin covalently bound to an enzyme capable of decomposing the 1,2-dioxetane compound with light energy emission, and a nylon membrane on which the nucleic acid or a fragment thereof is hybridized to the oligonucleotide probe. Such a kit in which R₁ is methoxy, R₂ is a meta-phosphate-substituted phenoxy group, and the enzyme is an acidic or alkaline phosphatase is preferred. The invention further relates to a kit for detecting a nucleic acid or a fragment thereof in a sample by hybridizing the nucleic acid or the fragment thereof with a complementarily labeled oligonucleotide probe. The kit comprises an enzymatically cleavable chemiluminescent 1,2-dioxetane compound as defined above, a complementary oligonucleotide probe covalently labeled with an antigen, an antibody directed against the antigen covalently bound to an enzyme capable of decomposing the 1,2-dioxetane compound with the emission of light energy, and a nylon membrane on which the nucleic acid or the fragment thereof is hybridized to the oligonucleotide probe.

A kit in which the enzyme is an acid or alkaline phosphatase and wherein R₁ is methoxy and R₂ is a meta-phosphate substituted phenoxy group is preferred.

The invention further relates to a kit for detecting a protein in a sample, which comprises an enzymatically cleavable chemiluminescent 1,2-dioxetane compound as defined above, an antibody directed against the protein covalently bound to an enzyme capable of decomposing the 1,2-dioxetane compound while emitting light energy, and a membrane on which the protein-antibody binding is carried out.

Preferably the membrane is a nylon, nitrocellulose or PVDF membrane.

According to a preferred embodiment of this kit, R1 is a methoxy group and R2 is a meta-phosphate substituted phenoxy group, and the enzyme is acid or alkaline phosphatase.

The invention further relates to a kit for detecting a protein in a sample, which comprises an enzymatically cleavable chemiluminescent 1,2-dioxetane compound as defined above, a first antibody directed directly against the protein and a second antibody directed against the first antibody, covalently bound to an enzyme capable of decomposing the 1,2-dioxetane compound.

According to a preferred embodiment of this kit, R1 represents a methoxy group and R2 represents a meta-phosphate substituted phenoxy group and the enzyme is an acid or alkaline phosphatase. It is desirable that the kit further comprises a water-soluble enhancing substance which increases the specific light energy production over that produced in its absence. Preferably, this enhancing substance is a polymeric quaternary ammonium salt, in particular poly(vinylbenzyltrimethylammonium chloride), poly[vinylbenzyl(benzyldimethylammonium chloride)] or poly[vinylbenzyl(tributylammonium chloride)].

According to a preferred embodiment, the invention relates to a kit in which the enhancing substance comprises a positively charged polymeric quaternary ammonium salt and fluorescein, which can form a ternary complex with the negatively charged product of the 1,2-dioxetane compound formed after enzyme-catalyzed decomposition of the 1,2-dioxetane compound, whereby energy transfer takes place between the negatively charged product and fluorescein and light energy is emitted by the fluorescein. The previously mentioned polymeric quaternary ammonium salts are preferably used in this kit.

Finally, the invention relates to a method for detecting an enzyme in a sample, which comprises the following steps:

(a) providing an enzymatically cleavable chemiluminescent 1,2-dioxetane compound as defined above;

(b) treating the 1,2-dioxetane compound with the sample containing the enzyme, whereby the enzyme cleaves the enzymatically cleavable labile substituent from the 1,2-dioxetane compound to form a negatively charged substituent bound to the 1,2-dioxetane compound, wherein the negatively charged substituent causes the 1,2-dioxetane compound to decompose to form a luminescent substance containing a fluorescent chromophoric group; and

(c) Detection of the luminescent substance as an indication of the presence of the enzyme.

This process involves the use of an acid or alkaline phosphatase as the enzyme and the use of a 1,2-dioxetane compound wherein R₁ is methoxy and R₂ is a me ta-phosphate-substituted phenoxy group is preferred. Also preferred is the use of galactosidase as the enzyme and a 1,2-dioxetane compound wherein R₁ is methoxy and R₂ is a meta-β-D-galactoside-substituted phenoxy group.

The following examples serve to further explain the invention to those skilled in the art. These examples are given for purposes of illustration only and do not constitute limitations except as set forth in the appended claims. All parts and percentages are expressed by weight per volume, except for TLC solution mixtures which are expressed by volume per volume, unless otherwise specified.

The ¹H NMR values reported in certain of the examples for the enol ether intermediates using a subscript symbol (') indicate aromatic protons, while numbers without a subscript in all cases indicate the substituent adamant-2'-ylidene ring positions, such as:

EXAMPLE I

200 g (1.64 mol) of 3-hydroxybenzaldehyde and 270 mL (1.93 mol) of triethylamine are added to a flask containing 1 L of methylene chloride in an ice bath. The resulting brown solution is stirred mechanically and 212 mL (1.72 mol) of trimethylacetyl chloride is added in a thin stream through a dropping funnel over 15 min. The resulting slurry is stirred for an additional 15 min, the ice bath is removed, and the reaction is then allowed to proceed for an additional 2 h. TLC (KSF; 25% acetone-hexanes) shows the absence of starting material and a single product of slightly higher Rf. The reaction mixture is transferred to a separatory funnel and mixed with 250 mL of 1M hydrochloric acid. The organic phase is then extracted with water (2 x 400 ml) and finally dried over sodium sulfate. The dried solution is passed through a layer of silica gel, evaporated on the rotary evaporator and then pumped under vacuum (1.0 mm Hg) to obtain 348 g of greenish-brown oil: 3-pivaloyloxybenzaldehyde, which is stored under argon atmosphere.

EXAMPLE 11

400 mg of p-toluenesulfonic acid dissolved in 25 ml of methanol are added to the 3-pivaloylbenzaldehyde of Example I with stirring.

Trimethyl orthoformate (224 mL, 2.05 mol) was then added dropwise. The slightly exothermic reaction was allowed to proceed uncontrolled while the mixture was stirred for 1 hour. ½ g of sodium bicarbonate was then added and the flask was placed on a rotary evaporator (bath temperature 40°C) to remove all volatiles. The resulting oil was passed through a short column of silica gel under nitrogen pressure to give an orange-brown oil which was pumped under vacuum (1 mm Hg) with stirring to give 426 g of crude 3-pivaloyloxybenzaldehyde dimethyl acetal. Infrared analysis showed no aldehyde carbonyl absorption (1695 cm-1).

EXAMPLE III

The crude 3-pivaloyloxybenzaldehyde dimethyl acetal from Example 11 is added to 1 L of methylene chloride freshly distilled from P2O5 in a 3 L flask under an argon atmosphere. Then 347 mL (2.03 mol) of triethyl phosphite is added all at once. The flask is fitted with a septum inlet adapter and is cooled in a dry ice-acetone bath under a low pressure of argon. Boron trifluoride etherate (249 mL, 2.03 mol) is added in several portions by syringe with vigorous stirring. The resulting reaction mixture is stirred at -55°C for 2 hours and then stored in a freezer at -20°C overnight.

The flask is then warmed to room temperature and its contents are stirred for 4 hours. The orange-brown solution is carefully added to a vigorously stirred slurry of 170 g of sodium bicarbonate in 800 mL of water at a rate to avoid violent foaming. After vigorously stirring the two-phase mixture for 1 hour, the layers are separated in a separatory funnel and the aqueous layer is again extracted with methylene chloride (2 x 250 mL). The combined organic extracts are dried over sodium sulfate, concentrated and distilled in vacuo to give 535 g of diethyl 1-methoxy-1-(3-pivaloyloxyphenyl)methanephosphonate as a clear, light yellow oil (b.p. 158-161°C at 0.25 mm Hg). This corresponds to a 91% yield for the overall procedure of Examples I to III:

1 H-NMR (400 MHz, CDCl3 ): ? 1.21 and 1.25 (6H, 2t, 7 Hz, OCH2 CH3 ), 3.37 (3H, s, ArCHOCH3 ), 3.80 (3H, s, ArOCH3 ), 3.90-4 .10 (4H, m, OCH2 CH3 ), 4.46 (1H, d, 15.6 Hz, ArCHPO), 6.85 (1H, m), 7.00 (2H, m), 7.26 (1H, m).

IR (pure): 2974, 1596, 1582, 1480, 1255 (P=O), 1098, 1050, 1020, 965 cm⁻¹.

EXAMPLE IV

A solution of diisopropylamine (11.6 mL, 82.8 mmol) in 75 mL of tetrahydrofuran was cooled to -78 °C in a dry ice-acetone bath under an argon atmosphere. 30 mL of 2.5 M solution of n-butyllithium in hexanes (Aldrich, 75.0 mmol) was added via syringe and after stirring the resulting lithium diisopropylamide solution for 20 minutes, 13.47 g (37.6 mmol) of diethyl 1-methoxy-1-(3-pivaloyloxyphenyl)methanephosphonate in 25 mL of tetrahydrofuran was added dropwise from a dropping funnel over 5 minutes. The resulting red solution is stirred at low temperature for a further 30 minutes to ensure complete formation of the phosphonate carbonate ion.

A solution of 4.99 g (30.1 mmol) of 5-hydroxyadamantan-2-one in 25 mL of tetrahydrofuran is then added dropwise. The resulting slightly cloudy mixture is stirred at -78°C for 5 min and slowly warmed to room temperature over 40 min. The solution, now orange in color, is heated at reflux for 50 min, cooled, diluted with 200 mL of saturated sodium bicarbonate solution and extracted with ethyl acetate (3 x 75 mL). The combined organic extracts were washed with a saturated sodium chloride solution, dried quickly over sodium sulfate and concentrated to give 12.49 g of an orange gum, a mixture of phenolic enol ether and its pivaloate ester.

1 H-NMR (pivaloate ester, 400 MHz in CDCl3 ): ? 7.33 (1H, m, H-5'), 7.12 (1H, J = 7.7 Hz, ArH), 6.95-7.02 (2H, m, ArH), 3.43 (1H , br. s, H-1), 3.28 (3H, s, OMe), 2.79 (1H, br. s, H-3), 2.23 (1H, br. s, H-7) , 1.59-1.87(11H, m); 1.34 (9H, s, COC(CH3 )3 ).

IR (in CHCl3 ): 3590, 3442 (OH), 2924, 2848, 1742 (ester C=O), 1665, 1602, 1578, 1426, 1274, 1152, 1118, 918 cm-1).

This mixture was taken up in methanol and heated to reflux for 3.5 hours in the presence of 10.7 g of anhydrous potassium carbonate. The methanol was then stripped off and the residue was partitioned between water and ethyl acetate. The organic layer was washed with saturated sodium chloride solution and rotary evaporated to a residue which was recrystallized from chloroform-petroleum ether to give 6.5 g of 3-(methoxy-5-hydroxytricyclo[3.3.1.13,7]deo-2-ylidenemethyl)phenol as a dull white solid (m.p. 171-172 °C). An additional 0.80 g was obtained from the mother liquor, giving an overall yield of phenolic enol ether based on 5-hydroxyadamantan-2-one of 79%.

1 H-NMR (400 MHz, in CDCl3 ): ? 7.18 (1H, dd, J = 8.4, 7.7 Hz, H-5'), 6.75-6.88 (3H, m, ArH), 6.36 (1H, br. s, ArOH), 3.41 (1H, br. s, H-1), 3.28 (3H, s, OMe), 2.79 (1H, br. s, H-3), 2.22 (1H, br.s, H-7), 1.56-1.98 (11H, m).

IR (in CHCl3 ): 3586, 3320 (OH), 3000, 2920, 2844, 1665, 1590, 1578, 1445, 1296, 1092, 885 cm-1.

HRMS calcd for C₁₈H₂₂O₃(M⁺) 286, 1573, found 286, 1569.

EXAMPLE V

A solution of 5.04 g (17.6 mmol) of 3-(methoxy-5-hydroxytricyclo[3.3.1.13,7]dec-2-ylidenemethyl)phenol in 35 mL of tetrahydrofuran, prepared under argon, was mixed with 3.4 mL (24.6 mmol) of triethylamine and then cooled to 0 °C in an ice bath. 2-Chloro-2-oxo-1,3,2-dioxaphospholane (1.95 mL, 21.1 mmol) was added dropwise with stirring. After 5 minutes, the ice bath was removed and stirring was continued for 45 minutes at room temperature. The reaction mixture was diluted with 30 mL of anhydrous diethyl ether and filtered under argon to exclude moisture. The triethylamine hydrochloride was then further ter with 20 ml of diethyl ether, the filtrate was concentrated on a rotary evaporator to give the phosphate triester as a viscous light orange oil.

The triester was dissolved in 30 mL of dimethylformamide dried over molecular sieves under argon and reacted with 1.02 g (20.8 mmol) of dry sodium cyanide added all at once for 3.5 hours at room temperature with stirring. The solvent was removed in vacuo (1.0 mm Hg) with heating to 50°C. A sample of the resulting orange-brown residue, after dissolving in water and performing reverse phase analytical chromatography [0.1% sodium bicarbonate (water) - acetonitrile gradient] on a PLRP polystyrene column (Polymer Laboratories), indicated complete conversion of the intermediate cyanoethyl phosphate diester sodium salt.

The residue was then taken up in 35 mL of methanol and treated dropwise with 4.85 mL of a 4.37 M solution (21.2 mmol) of sodium methoxide in methanol with stirring for 30 min at room temperature. Reverse phase analytical HPLC showed that β-elimination was complete for the phosphate monoester. The solvent was removed and the residue was triturated with 10% water/acetone to give a gummy solid. Further trituration with 3% water/acetone gave a hard, almost dull white solid which was filtered and dried in vacuo (1.0 mm Hg) to give 8.35 g of crude disodium 3-(methoxy-5-hydroxytricyclo[3.3.1.13,7]dec-2-ylidenemethyl)phenyl phosphate contaminated with inorganic salts.

Preparative reversed-phase HPLC using a water-acetonitrile gradient on a PLRP polystyrene column (Polymer Laboratories) and lyophilization of the appropriate fractions gave 5.2 g (72%) of purified compound as a white granular solid which softened at 120 °C and melted to a light brown gum at 163-168 °C.

1 H NMR (400 MHz, in D2 O). &delta; 7.16 (1H, dd, J = 8.3, 7; 4 Hz, H-5'), 7.05 (1H, d, J = 8.3 Hz, ArH), 6.93 (1H, br .s, H-2'), 6.86 (1H, d; J = 7.4 Hz, ArH), 3.2 (3H, s, OMe), 3; 17 (1H, br. s, H-1), 2.61 (1H, br. s, H-3), 2.06 (1H, br. s, H-7), 1.42-1.73 (IOH, m).

Elemental analysis showed that the phosphate salt was present as a dihydrate. Analysis calculated for C18H21Na2O6P·2H2O: C, 48.44, H, 5.65, P, 6.94. Found: C, 48.37, H, 5.90, P, 6.87.

EXAMPLE VI

A solution of 0.8 g of disodium 3-(methoxy-5-hydroxytricyclo[3.3.1.13,7]dec-2-ylidenemethyl)phenyl phosphate in 96 ml of 25% anhydrous methanol/chloroform containing 5.35 x 10-5 M methylene blue sensitizing dye was dispensed into three glass test tubes. Each tube was then cooled to 5°C in a water bath and saturated with oxygen by passing a gas stream through the solution. After 5 minutes, while oxygen was still bubbling through the solution, the tubes were irradiated with light from a 250 watt high pressure sodium vapor lamp while maintaining the temperature at 5°C. A 5 mil thick piece of Kapton polyimide film (duPont) was placed between the sodium vapor lamp and the tubes to filter out unwanted UV radiation. After 20 minutes of irradiation After chromatography, the solutions had turned pink. Analytical reversed-phase HPLC [0.1% sodium bicarbonate (water - acetonitrile gradient)] on a PLRP polystyrene column (Polymer Laboratories) showed two product peaks: an early-eluting broadened peak (retention time 3.79 minutes) and a later-eluting sharp peak (retention time 5.77 minutes). The ratio of the earlier-eluting product (A) to the later-eluting product (B) was 1.3:1 in each tube solution.

The solutions were combined and the solvent was removed in vacuo (25.0 mm Hg) on ice bath. The residue was dissolved in 70 mL of water and filtered through a 0.45 µm nylon membrane. Reverse-phase preparative HPLC (water-acetonitrile gradient) easily separated two products.

Combination of the corresponding fractions from preparative HPLC followed by analytical HPLC showed that they were homogeneous. Lyophilization gave 0.32 g of one product (A) and 0.26 g of another product (B) which were shown by 1H NMR to be isomers (syn and anti) of 1,2-dioxetanes, namely syn-disodium 3-(4-methoxyspiro-(1,2-dioxetane-3,2'-(5'-hydroxy)tricyclo[3.3.1.13,7]decan)-4-yl)phenyl phosphate:

and its anti-isomer (anti-disodium-3-(4-methoxyspiro-[1,2-dioxetane-3,2'-(5'-hydroxy)-tricyclo[3.3.1.13,7]decan]-4-yl )phenyl phosphate):

Each of these isomers produced chemiluminescence with unique light (wavelength) versus time and noise profiles upon cleavage at pH 10 in aqueous buffer medium containing alkaline phosphatase.

1 H NMR (A isomer, 400 MHz in D2 O): ? 6.98-7.6 (4H, m, ArH), 3.11 (3H, s, OMe), 2.97 (1H, br. s, H-1), 2.34 (1H, br. s , H-3), 1.79 (1H, br. s, H-7), 1.3-1.68 (8H, m), 1.01 (1H, d,J = 13.5 Hz), 0.9(1H, d,J = 13 Hz).

1 H NMR (B isomer, 400 MHz in D2 O): ? 6.94-7.62 (4H, m, ArH), 3.09 (3H, s, OMe), 2.91 (1H, br. s, H-1), 2.27 (1H, br. s , H-3), 1.84 (1H, br. s, H-7), 1.21-1.75 (8H, m), 1.02 (1H, d, J = 12.8 Hz), 0.87 (1H, d, J = 12.8 Hz).

Elemental analysis showed that isomer B was present as the dihydrate. Analysis calculated for C₁₈H₂₂Na₂O₈P·2H₂O (isomer B): C, 45.2, H, 5.27, P, 6.47. Found: C, 45.53, H, 5.35, P, 6.11.

Based on the ¹H NMR data, it was not possible to specifically determine which of the two obtained isomers was the syn isomer and which was the anti isomer.

EXAMPLE VII

The procedure of Example IV above was generally followed. A solution of 5.35 mL (38.2 mmol) of diisopropylamine in 35 mL of tetrahydrofuran was cooled to -78°C in an ice bath under an argon atmosphere. 14 mL of a 2.5 M solution of n-butyllithium in hexanes (35.0 mmol) was added by syringe and after stirring for 20 minutes, 10.86 g (30.3 mmol) of diethyl 1-methoxy-1-(3-pivaloyloxyphenyl)methanephosphonate in 30 mL of tetrahydrofuran was added dropwise over 10 minutes. The resulting orange solution was stirred at low temperature for 1 hour, then mixed with a solution of 5.21 g (22.75 mmol) of 5-bromoadamantan-2-one in 20 mL of tetrahydrofuran over 7 minutes with stirring. Stirring was continued for 10 minutes. At this time, the cold bath was removed and the reaction mixture was allowed to warm slowly to room temperature over 1 hour.

The solution was then heated at reflux for an additional 1 hour, cooled, diluted with 100 mL of hexanes, and transferred to a separatory funnel containing a saturated aqueous sodium bicarbonate solution and extracted with 10% ethyl acetate in hexanes (3 x 50 mL). The combined organic extracts were washed with 15% aqueous sodium chloride solution, dried quickly over sodium sulfate, and concentrated to give 11.62 g of a light orange viscous oil. Plug filtration on a short silica gel column eluting with 10% ethyl acetate in n-hexanes gave 11.0 g of a yellowish-green gum.

1 H-NMR (pivaloyl ester, 400 MHz in CDCl3 ): ? 7.34 (1H, t, J = 7.8 Hz, H-5'), 7.1 (1H, d, J = 7.7 Hz, ArH), 6.95-7.02 (2H, m , ArH), 3.39 (1H, br. s, H-1), 3.28 (3H, s, OMe), 2.74 (1H, br. s, H-3), 2.32-2 .51 (6H, m, H-4, H-6, H-9), 2; 17 (1H, br. s, H-7), 1.67-1.92 (4H, m, H-8, H-10), 1.34 (9H, s, COC(CH3 )3 ) :

IR (pure): 2924, 2850, 1745 (ester C=O), 1654, 1602, 1578, 1478, 1274, 1110, 1018, 808, 758 cm⁻¹.

This gum was taken up in methanol and refluxed with 2.4 g of anhydrous potassium carbonate for 2 hours. The methanol was then removed and the residue was partitioned between water and 30% ethyl acetate in hexanes. The organic layer was washed with an aqueous saturated sodium chloride solution, dried over sodium sulfate and concentrated to give 9.32 g of a yellow gum. The gum was flash chromatographed on silica gel to give 7.06 g (88% yield based on 5-bromoadamantan-2-one) of a yellowish gum which could not be crystallized: however, IR and NMR indicated that the resulting compound was 3-(methoxy-5-bromotricyclo[3.3.1.13,7]dec-2-ylidenemethyl)phenol, which was pure enough to be used in the phosphorylation reaction.

A sample of the pure phenolic compound was obtained from 3-(methoxy-5-bromotricyclo[3.3.1.13,7]dec-2-ylidenemethyl)phenylacet as follows: the impure phenolic compound (5.57 g, 15.95 mmol) was dissolved in 30 mL of pyridine dried over molecular sieves under argon atmosphere. 50 mg of 4-dimethylaminopyridine was added as a catalyst. Then 1.8 mL (19.15 mmol) of acetic anhydride was added via syringe and the reaction mixture was stirred at room temperature for 3 hours.

The reaction mixture was then transferred to a separatory funnel containing 250 mL of aqueous saturated sodium bicarbonate solution and then extracted with 10% ethyl acetate in hexanes (2 x 100 mL). The combined extracts were washed several times with water and then dried quickly over sodium sulfate. The dried solution was concentrated in a rotary evaporator and the residue was recrystallized from n-hexane containing a few drops of ethyl acetate. The resulting dull white solid was recrystallized again to give 5;21 g (83.5% yield) of the acetate, mp 108-110 °C.

HRMS calcd for C20H23BrO3(M+) 390.0833, found, 390.0831.

1 H-NMR (400 MHz in CDCl3 ): ? 7.35 (1H, dd, J = 8, 7.7 Hz, H-5'), 7.13 (1H; dd, J = 7.7, 1 Hz, ArH), 7.03 (1H, dd , J = 8, 1 Hz, ArH), 5.99 (1H, d, 1 Hz, H-2'), 3.39 (1H, br. s, H- 1), 3.27 (3H, s , OMe), 2.75 (1H, br. s, H-3), 2.32-2.51 (6H, M, H-4, H-6, H-9), 2.29 (3H, s, OAc), 2.18 (1H, br. s, H-7), 1.69-1.92 (4H, m, H-8, H-10).

IR (in CHCl3 ): 3000, 2930, 2850, 1760 (ester C=O), 1660, 1602, 1577, 1868, 1192, 1095, 1070, 1016, 804 cm-1.

Treatment of the recrystallized acetate with potassium carbonate in methanol for 15 hours at room temperature gave 3-(methoxy-5-bromotricyclo[3.3.1.13,7]dec-2-ylidenemethyl)phenol, m.p. 42-45 °C, as a white, friable foam which could not be further recrystallized.

1 H-NMR (400 MHz in CDCl3 ): ? 7.21 (1H, t, J = 7.2 Hz, H-5'), 6.73-6.85 (3H, m, ArH), 5.18 (1H, s, ArOH), 3.38 (1H, br. s, H-1), 3.28 (3H, s, OMe), 2.74 (1H, br. s, H-3), 2.3- 2.52 (6H, m, H-4, H-6, H-9), 2.17 (1H, br. s, H-7), 1.68-1.92 (4H, m, H-8, H-10).

IR (in CHCl3 ): 3584, 3320 (OH), 2925, 2850, 1665, 1588, 1578, 1445, 1435, 1092, 1080, 1015, 880, 808 cm-1.

EXAMPLE VIII

The general procedure of Example IV above was again followed. A solution of 2.97 mL (21.3 mmol) of diisopropylamine in 21 mL of tetrahydrofuran was cooled to -78°C in a dry ice-acetone bath and under an argon atmosphere, mixed with 8.5 mL of a 2.5 M solution of n-butyllithium in hexanes (21.3 mmol) added dropwise by syringe, and stirred for 20 minutes. Diethyl 1-methoxy-1-(3-pivaloyloxyphenyl)methanephosphonate (7.26 g, 20.3 mmol) in 20 mL of tetrahydrofuran was then added dropwise by syringe over 10 minutes, and the solution was stirred at low temperature for 1 hour.

A solution of 2.79 g (15.2 mmol) of 5-chloroadamantan-2-one in 15 mL of tetrahydrofuran was added over 5 minutes and after stirring at low temperature for 10 minutes, the cold bath was removed and the mixture warmed to room temperature. The mixture was then heated at reflux for 1.5 hours, cooled, diluted with 50 mL of n-hexanes and poured into a separatory funnel containing 150 mL of an aqueous saturated sodium bicarbonate solution. Extraction with 5% ethyl acetate-n-hexane was followed by drying the combined organic fractions, concentrating them and pumping in vacuo (1.0 mm Hg) to give a residue which was chromatographed on silica gel to give 5.15 g of the higher Rf 3-(methoxy-5-chlorotricyclo[3.3.1.13,7]dec-2-ylidenemethyl)phenyltrimethylacetate as a colorless oil (structure confirmed by IR and NMR) and 0.865 g of late eluting material composed mainly of the corresponding phenol. This latter fraction was reacylated with pivaloyl chloride and triethylamine in methylene chloride (see Example I above) and then chromatographed to give an additional 0.35 g of pivaloyl ester (93% overall yield based on 5-chloroadamantan-2-one).

1 H-NMR (pivaloyl ester, 400 MHz, in CDCl3 ): ? 7.36 (1H, t, J = 7.8 Hz, H-5'), 7.13 (1H, d, J = 7; 7 Hz, ArH), 6.98-7.04 (2H, m , ArH), 3.45 (1H, br. s, H-1), 3.3 (3H, s, OMe), 2.8 (1H, br. s, H-3), 2.13-2 .32 (7H, m, H-4, H-6, H-7, H-9), 1.65-1.9 (4H, m, H-8, H-10), 1.36 (9H , s, COC(CH3 )3 ).

IR (pure): 2932, 2835, 1750 (ester C=O), 1664, 1602, 1578, 1478, 1274, 1112, 1022, 827, 758 cm⁻¹.

The combined pivaloyl ester fractions (5.5 g, 14.1 mmol) were taken up in 40 mL of methanol and heated to reflux with 5.37 g of anhydrous potassium carbonate for 40 minutes. The residue remaining after stripping off the methanol was partitioned between water and 30% ethyl acetate-hexanes, and the organic fractions were concentrated and plug filtered on a short silica gel column to give 4.07 g (88% yield based on 5-chloroadamantan-2-one) of 3-(methoxy-5-chlorotricyclo[3.3.1.13,7]dec-2-ylidenemethyl)phenol as a friable white foam which became slightly sticky on exposure to air.

HRMS calcd for C₁₈H₂₁ClO₂(M⁺) 304, 1227, found 304, 1230.

1 H-NMR (400 MHz, in CDCl3 ): ? 7.23 (1H, J = 7.7, 7.6 Hz H-5') 6.85d, J = 7.6 Hz, ArH), 6.77-6.83 (2H, m, ArH), 3.44 (1H, br. s, H-1), 3.31 (3H, s, OMe), 2.8 (1H, br. s, H-3), 2.1-2.31 (7H , m, H-4, H-6, H-7, H-9), 1.65-1.89 (4H, m, H-8, H-10).

IR (in CHCl3 ): 3590, 3330 (OH), 2930, 2855, 1655, 1590, 1580, 1440, 1295, 1094, 1080, 1022, 880, 826 cm-1.

EXAMPLE IX

3-(Methoxy-5-bromotricyclo[3.3.1.13,7]dec-2-ylidenemethyl)phenol (1.49 g, 4.26 mmol) was dissolved in 8.5 mL of anhydrous ethylene glycol and then added to a tightly sealed glass tube with 0.28 g of potassium carbonate. The tube was heated in an oil bath at 110 °C for 7 h. The contents were then concentrated in vacuo (1.0 mm Hg) with heating. The residue was partitioned between saturated sodium chloride solution and ethyl acetate. The organic fraction was then stripped and chromatographed on a short column of silica gel to give 1.31 g (92% yield based on the phenol starting material) of 3-(methoxy-5-(2-hydroxy)ethoxytricyclo[3.3.1.1.3,7]dec-2-ylidenemethyl)phenol as a dull white foam.

1 H-NMR (400 MHz, in CDCl3 ): ? 7.19 (1H, t, J = 7.6 Hz, H-5'), 6.83 (1H, d, J = 7.6 Hz, ArH), 6.73-6.80 (2H, m , ArH), 5.83 (1H, s, ArOH); 3.67 (211, m, OCH2 CH2 OH), 3.51 (2H, t, J = 4.6 Hz, OCH2 CH2 OH), 3.44 (1H, br. s, H-1 ), 3.28 (3H, s, OMe), 2.81 (1H, br. s, H-3), 2.24 (1H, br. s, H-7), 1.55-1.90 (10H, m).

IR (CHCl3 ): 3580, 3320 (OH), 2929, 2842, 1664, 1586, 1575, 1440, 1092, 1078, 885 cm-1.

EXAMPLE X

3-(Methoxy-5-chlorotricyclo[3.3.1.1.3,7]dec-2-ylidenemethyl)phenol (1.23 g, 4.0 mmol) was phosphorylated in the same manner as described in Example V above with one exception - ammonia in methanol was used for β-elimination. The resulting crude ammonium sodium salt was triturated with acetone, then pumped in vacuo (1.0 mm Hg) to give 1.2 g of dull white solid. Analytical reverse-phase HPLC showed that the obtained phosphorylated enol ether product was pure enough for direct photooxygenation to the corresponding 1,2-dioxetane.

3-(Methoxy-5-chlorotricyclo[3.3.1.1.3,7]dec-2-ylidenemethyl)phenyl phosphate salt (0.65 g) was dissolved in 100 mL of anhydrous 10% methanol/chloroform also containing 5.35 x 10-5 M methylene blue as a sensitizing dye. The resulting solution was divided into three glass test tubes and irradiated as described in Example VI above. The preparation Processing in this case involved dissolving the pumped residue in 70 mL of water containing 258 mg of sodium bicarbonate. When performing preparative reversed-phase HPLC, syn and anti isomers were collected together, excluding impurities. Analytical HPLC [0.1% NaHCO3(H2O)-acetonitrile gradient] showed two product peaks (retention times 8.01 and 8.32 minutes). The area percent ratio (270 nm) of the first eluted isomer to the later eluted isomer was 0.4:1. 1H NMR confirmed that the resulting lyophilized white solid was a mixture of syn- and anti-disodium 3-(4-methoxyspiro-[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1.3,7]decan-2-yl)phenyl phosphate.

Elemental analysis indicated that the product exists as a dihydrate. Analysis calculated for C18H20ClNa2O7P·2H2O: C, 43.52, H, 4.87, Cl, 7.14. Found: C, 43.23, H, 4.99, Cl, 7.65.

1H NMR (400 MHz, in D2O, two isomers): δ 6.97-7.68 (4H, m, ArH), 3.08 and 3.09 (3H, 25, OMe), 2.95 (1H, br. s, H-1), 0.76-2.35 (12H, m).

EXAMPLE XI

3-(Methoxy-5-bromotricyclo[3.3.1.13,7]dec-2-ylidenemethyl)phenol was phosphorylated and then photooxygenated as described for the 5-chloro analogue in Example X above. Work-up in 0.3% (w/v) aqueous sodium bicarbonate solution afforded a filtered aqueous solution of the crude 1,2-dioxetane phosphate salt, which was then subjected to preparative reverse-phase HPLC (water-acetonitrile gradient) to yield the syn and anti isomers, which were collected together for lyophilization. When the lyophilized product, a white, flaked solid, was subjected to analytical HPLC, two peaks with retention times of 8.52 and 8.94 minutes were obtained. The area percent ratio (270 nm) of the first eluting isomer to the later eluting isomer was 0.5:1. The 1H NMR confirmed that the product was a mixture of syn- and anti-disodium 3-(4-methoxyspiro-[1,2-dioxetane-3,2'-(5'-bromo)tricyclo[3.3.1.1.3,7]decan-4-yl)phenyl phosphate.

1H NMR (400 MHz, in D2O, two isomers): δ 6.99-7.52 (4H, m, ArH), 3.07 and 3.09 (3H, 25, OMe), 2.91 (1H, br. s, H-1), 0.822.32 (12H, m).

EXAMPLE XII

Immunoassays for TSH were performed on a panel of TSH standards using a Hybritech Tandern-E TSH kit (Hybritech, Inc., San Diego, California) according to the manufacturer's instructions provided with the kit, except that after completion of the anti-TSH alkaline phosphatase conjugate incubation step and washing, the plastic beads were additionally washed with 0.1 M diethanolamine, 1 mM magnesium chloride, 0.02% sodium azide buffer, pH 10.0, and then stored briefly in 200 μl of the same buffer. Chemiluminescence signals from anti-TSH-alkaline phosphatase conjugate bound to the surface of the beads were detected by adding to the tubes containing beads 0.67 mM buffer solutions containing disodium 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane ("AMPPD"), Disodium 3-(4-methoxyspiro-[1,2-dioxetane-3,2'-(5'-hydroxy)tricyclo[3.3.1.1.3,7]decan]-4-yl)phenyl phosphate (A isomer; "A OH-AMPPD"), the corresponding disodium B isomer ("B-OH-AMPPD") and disodium 3-(4-methoxyspiro-[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1.3,7]decan]-4-yl)phenyl phosphate ("Cl-AMPPD") in 0.1 M diethanolamine, 1 mM magnesium chloride, 0.02% sodium azide, pH 10.0. The intensity of light emission was measured sequentially at 7, 13, 19, 25, 31, 40, 50 and 60 minutes after substrate addition as a 5 second integral at room temperature (approximately 25ºC) using a Berthold LB952T luminometer (Berthold Instrument, Wildbad, Federal Republic of Germany). (Only the signals, no signal-to-noise were measured).

TSH, RLU v. TSH for each AMPPD, BR-AMPPD, B-OH-AMPPD, A-OH-AMPPD and CL-AMPPD are shown in Figures 1, 2, 3, 4 and 5, respectively.

EXAMPLE XIII

A comparison of the total luminescence emission of AMPPD and the corresponding 1,2-dioxetanes whose adamant-2'-ylidene groups were substituted with hydroxy (A and B isomers), chlorine and bromine groups was performed by performing total dephosphorylation experiments on each of these compounds.

An aqueous solution of the 1,2-dioxetane (4.0 mM) in 0.05 M sodium carbonate/sodium bicarbonate containing 1 mM magnesium chloride was prepared and then equilibrated at 30°C. 10 μl of a 7.64 x 10 M aqueous solution of alkaline phosphatase (calf intestine; Biozyme) was then added and the chemiluminescence of the resulting solution was measured using a Turner Model 20E luminometer (Turner Instruments; Sunnyvale, California).

The rates of chemiluminescence decay for each of the five compounds in question, expressed as relative light units (RLU) per minute, are given in the following table. Table I Decay rate (RLU/minute)

These total chemiluminescence emissions are graphically presented in Figs. 6, 7, 8, 9 and 10, respectively.

EXAMPLE XIV

A strip of neutral BIODYNE A nylon membrane (Pall Corporation, Glen Cove, NY) was coated twice (side by side) with the following concentrations of biotinylated pBR-322 35-mer oligonucleotide probe (Synthetic Genetics, San Diego, California):

1/ Single-stranded, unlabeled DNA, 1 ng.

The membrane was then blocked in 0.2% casein/0.1% Tween 20 detergent in aqueous phosphate buffered saline (PBS) for 1 hour, after which 1/5000 diluted avidin-alkaline phosphatase conjugate (Sigma, Inc., St. Louis, MO) in 0.2% casein in PBS was added. The membrane was then incubated for 30 minutes, washed twice (5 minutes each) in 0.2% casein/0.1% Tween 20 detergent in PBS, washed four times (5 minutes each) in 0.3% Tween 20 detergent in PBS, and washed twice (5 minutes each) in aqueous 0.1 M diethanolamine containing 1 mM magnesium chloride, pH 10.0.

The membrane was then cut in half to yield two strips, each containing one set of dots. One of the strips was incubated for 5 minutes in aqueous AMPPD solution (0.25 mM in 0.1 M diethanolamine containing 1 mM magnesium chloride, pH 10), the other was incubated for 5 minutes in the corresponding chloroadamant-2'-ylidene compound (0.25 mM in the same buffer). The two strips were then placed in camera-luminometers and exposed with Polaroid Type 612 instant black and white film. The enhanced chemiluminescence intensity obtained using the chloro compound compared to AMPPD itself is evident when comparing column 2 (C1-AMPPD) with column 1 (AMPPD) in Figure 11.

EXAMPLE XV

pBR 322 plasmid (4700 bp) was subjected to a nick translation procedure using a Trans-Light kit (Tropix, Inc., Bedford, Mass.) to generate a mixture of biotinylated single-stranded polynucleotides ranging from 200 to 2000 bp in length. This mixture was then spotted onto a dry BIODYNE A membrane as five parallel columns of spots of the following concentrations.

The membrane was subjected to ultraviolet irradiation (UVP Mineral Light; UVP, San Gabriel, Calif.) for 3 minutes to fix the DNA to the surface of the membrane and then air dried. The membrane was then blocked in 0.2% casein/0.1% Tween 20 detergent in PBS for 1 hour, after which 1/5000 diluted avidin-alkaline phosphatase conjugate (Tropix, Inc.) in 0.2% casein in PBS was added. The membrane was then incubated for 30 minutes, washed three times (5 minutes each) in 0.2% casein/0.1% Tween 20 detergent in PBS, and washed once for 5 minutes in aqueous 0.1 M diethanolamine containing 1 mM magnesium chloride and 0.02% sodium azide, pH 10.0 (substrate buffer).

Thereafter, the five columns of rows of spots 1-5 were individually cut out of the membrane ("strips 1-5"), as were the five columns of rows of spots 6-10 ("strips 6-10"). Strips 1-5 were washed with substrate buffer for 30 minutes. Strips 6-10 were blocked in 0.1% BDMQ in substrate buffer for 30 minutes. Both sets of strips were then incubated for 5 minutes in substrate buffer, then individually for 5 minutes in aqueous solutions (0.25 mM) of 1,2-dioxetanes as indicated below:

All strips were then placed in camera luminometers and exposed to Polaroid Type 612 instant black and white film. The improved chemiluminescence intensity obtained using 3-(substituted adamant-2'-ylidene)-1,2-dioxetanes compared to AMPPD itself is evident from the results in Table 11 below. Table II ALKALINE PHOSPHATASE LABELLED DNA PROBE DETECTION IN A MEMBRANE WITH AND WITHOUT BDMQ BLOCKING STAGE

1/ A 5-minute exposure was carried out 40 minutes after dioxetane addition

2/ A 1-minute exposure was carried out 77 minutes after dioxetane addition

EXAMPLE XVI

On a strip of neutral BIODYNE-A nylon membrane, 12.5 picograms of biotinylated pBR 322 35-mer oligonucleotide probe (Biogen, Inc., Cambridge, Mass.) were spotted twice (side by side); dried and subjected to ultraviolet irradiation using a UVP mineral light lamp for 5 minutes to fix the DNA to the surface of the membrane. The membrane was then wetted with 5 X SSC (0.015 M sodium citrate/0.15 M sodium chloride) and blocked in 0.2% casein, 0.1% Tween 20 detergent in PBS for 1 hour. The blocked membrane was then incubated with avidin-alkaline phosphatase conjugate (“Avidx” conjugate; Tropix, Inc.) diluted 1-10,000 in 0.2% casein for 30 minutes.

The membrane was then washed twice (5 minutes each) with aqueous 0.2% casein/0.1% Tween 20 detergent, then twice (5 minutes each) with aqueous 0.1% Tween 20 detergent in PBS, and finally twice (5 minutes each) in aqueous diethanolamine containing 1 mM magnesium chloride, pH 10 (assay buffer).

The washed membrane was cut in half to obtain two strips, each bearing a dot. The strips were then incubated in 0.25 mM aqueous solutions of AMPPD and the corresponding chloroadamant-2'-ylidene compound in 0.1 M diethanolamine containing 1 mM magnesium chloride, pH 10, for 5 minutes, then drained and sealed in plastic bags that were taped to the window of a Turner Model 30E luminometer. The chemiluminescence emission from each strip was integrated for 20 hours. The kinetic values for the obtained light emissions are shown in Figure 12.

EXAMPLE XVII

The enhancement of the chemiluminescence emission (compared to the emission of AMPPD) obtained from the corresponding hydroxyadamant-2'-ylidene (A and B isomers) and chloroadamant-2'-ylidene compounds, all in the further presence of enhancer polymers given below, is demonstrated in the following manner.

Four sets of three tubes, each tube in each set containing 450 μl of a 0.4 mM aqueous solution of one of the four 1,2-dioxetanes compared in 0.1 M diethanolamine containing 1 mM magnesium chloride, 0.02% sodium azide and 0.1% enhancement polymer, pH 10.0 (substrate buffer), were prepared and the background signal from each tube, determined using a Berthold LB 952T luminometer (Berthold Instruments, Wildbad, Federal Republic of Germany), was measured.

Thereafter, 50 µl of an aqueous solution 2.83 x 10-12 M of alkaline phosphatase in 0.1 M diethanolamine containing 1 mM magnesium chloride, 0.02% sodium azide, pH 10.0, (final enzyme concentration 2.83 x 10-13 M) was added to each tube and the chemiluminescence signals were measured in the luminometer at 5 and 20 minutes. The intensity of the chemiluminescence signals and the signal:background ratios obtained for each of the four 1,2-dioxetanes in the presence of enhancing polymers are given in Table 3 below.

The reinforcing polymers used and the symbols for each of the polymers used in Table III are as follows: TABLE III

EXAMPLE XVIII

The background signals and t1/2 parameters (compared to those of AMPPD) obtained for the substituted adamant-2'-ylidene compounds given below were determined in two different buffers in the following manner.

Aqueous 4 x 10-4 M solutions of 1,2-dioxetanes in a buffer solution prepared from 0.05 M sodium carbonate/sodium bicarbonate containing 1 mM magnesium chloride, pH 9.5, were prepared, as were aqueous 4 x 10-4 M solutions of the 1,2-dioxetanes in a buffer solution prepared from 0.1 M diethanolamine containing 1 mM magnesium chloride and 0.02% sodium azide, pH 10.0. 1 ml of each of these solutions per tube was placed in a Turner Model 20E luminometer and background signals were determined.

Thereafter, t1/2 values were measured for each sample as follows. 100 µl of the sample dioxetane in 900 µl of one of the buffer solutions described above was pipetted into a test tube (final dioxetane concentration 4 x 10-5 M) and equilibrated at 30°C. 10 µl of a 1 to 1000 dilution of calf intestinal alkaline phosphatase in the same buffer (enzyme concentration 7.6 x 10-10 M) was added and the resulting chemiluminescence intensity was recorded using a Turner Model 20E luminometer over 30 minutes. t1/2 values were then calculated from the decay curves. The results of these determinations are given in Table IV below. TABLE IV 1. 0.05 M sodium carbonate/sodium bicarbonate, 1 mM magnesium chloride, pH 9.5 2. 0.1 M diethanolamine, 1 mM magnesium chloride, 0.02% sodium azide, pH 10.0

EXAMPLE XIX

Alkaline phosphatase (calf intestine; Biozyme) was diluted to 1-1,000,000 to produce a stock solution (concentration 2.54 x 10-11 M). A series of test tubes were prepared in duplicate containing 450 µl of an aqueous 0.1 M diethanolamine solution containing 1 mM magnesium chloride and 0.02% sodium azide, pH 10, plus 0.1% of one of the reinforcing polymers indicated below, and 4.4 x 10-4 M of a 1,2-dioxetane: AMPPD or the corresponding chloroadamant-2'-ylidene compound.

50 μl of alkaline phosphatase stock solution was then added to give samples with the following enzyme concentrations:

The final concentration of 1,2-dioxetane in each tube was 4 x 10-4 M and the final concentration of enhancer polymer was 0.09%. Five second integrals were recorded at 5 and 20 minutes after 1,2-dioxetane addition.

Figure 13 shows the dose response curves for alkaline phosphatase dilution with chloradamant-2'-ylidene-1,2-dioxetane with BDMQ 5 minutes after dioxetane addition. Figure 14 shows the alkaline phosphatase dilutions detected with chloradamant-2'-ylidene-1,2-dioxetane plus BDMQ 20 minutes after substrate addition compared with AMPPD plus BDMQ.

EXAMPLE XX

The pBR322 plasmid (Biogen, Inc., Cambridge, Mass.) containing an insert of a TPA sequence was digested with MSP1 restriction enzyme. Chemical digestions were performed as described by Maxam, et al., PNAS. 74 560 (1977), yielding G, AG, AC, TC, and C - and another, T, as described by Rubin, et al., Nucleic Acids Research. 8 4613 (1980). One seventh of each reaction tube content was loaded per row onto a 0.4 mm TBR gradient sequencing gel (60 cm length). After 4 hours of electrophoresis, the DNA was transferred to a BIODYNE A nylon membrane and treated with ultraviolet light to fix the DNA to the membrane surface.

The membrane was then dried, prehybridized for 30 minutes at 45ºC in aqueous buffer solution containing 1% BSA, 0.5 M sodium phosphate and 7% SDS, pH 7.2, then incubated for 2 hours at 45ºC with 10 µl NNB snap direct alkaline phosphatase conjugated probe (Molecular Biosystems, Inc., San Diego, California) in 40 ml of the above. The membrane was then washed twice in aqueous 5 X SSCh% SDS (for 5 minutes each) at 45ºC, twice in aqueous 1 X SSC/1% SDS (for 5 minutes each) at 45ºC, once in an aqueous solution containing 125 mM sodium chloride, 50 mM Tris and 1% Triton X-100 detergent, pH 8.0, twice in aqueous 1 X SSC (for 1 minute each) at room temperature and finally twice (for one minute each) at room temperature in an aqueous solution containing 0.1 M diethanolamine, 1 mM magnesium chloride and 0.02% sodium azide, pH 10.0.

The membrane was then wetted with aqueous AMPPD solution (0.25 mM), wrapped in Saran wrap and exposed to Kodak XAR X-ray film for 40 min. A 5 min exposure was then performed 1 h after AMPPD addition. The sequence images are shown in Fig. 17(1).

Repeating this entire procedure using the corresponding chloroadamant-2'-ylidene-1,2-dioxetane compound yielded the sequence images shown in Fig. 17(2).

EXAMPLE XXI

The thermal background of each dioxetane, as indicated in Table 4, was measured in a solution of 0.4 mM dioxetane in 0.1 M diethanolamine, 1 mM MgCl2, pH 10.0, at 30°C in a Turner Model 20E Luminometer (Sunnyvale, CA). The average chemiluminescence intensity of each sample in the absence of enzyme is listed under "Background at 0.4 mM in Turner Light Units (TLU)" in Table 4.

The half-life of the dioxetane anion, also given in Table 4, was measured as follows: a 1 ml solution of 0.04 mM dioxetane (in the same buffer as above) was completely dephosphorylated by the addition of 0.764 picomoles of alkaline phosphatase at 30ºC in a Turner Model 20E luminometer. The half-life of the dioxetane anion was then calculated from the first order decay of the chemiluminescence emission. TABLE 4 COMPARISON OF HALF-LIFE AND THERMAL BACKGROUNDS OF VARIOUS DIOXETONE PHOSPHATES

TLE = Turner Light Units

* Anion prepared by adding 0.764 picomoles of alkaline phosphatase to 40 nanomoles of AMPPD and R-AMPPD in 0.1 M DEA, 1 mM MgCl2, pH 10.0.

EXAMPLE XXIII

The detection capabilities of alkaline phosphatase with different chemiluminescent 1,2-dioxetane substrates were determined as follows: duplicates of serial dilutions (1 to 3) of alkaline phosphatase were incubated at room temperature with 0.4 mM dioxetane in 0.1 M diethanolamine, 1 mM MgCl2, pH 10.0, containing 1 mg/ml of either polymeric enhancer 1 or enhancer 2 in a Berthold LB952T luminometer (Wildbad, Germany).

Following either a 5 minute or 20 minute incubation with a substrate, the chemiluminescence intensity was determined as 5 second integrated relative light units (RLU) and plotted graphically as shown in Figure 18. The alkaline phosphatase concentration that gave a chemiluminescent signal twice the signal obtained without the enzyme was extrapolated from the graph of Figure 18. The alkaline phosphatase concentrations at twice the background were used as detection limits in Table 5. TABLE 5 DETECTION LIMITS FOR ALKALINE PHOSPHATASE WITH DIFFERENT CHEMILUMINESCENCE 1,2-DIOXETONE SUB STRATE/AMPLIFIER SYSTEMS

The detection limits are given in femtomol/liter.

EXAMPLE XXIII

Duplicates of serial dilutions (1 to 3) of alkaline phosphatase were incubated at room temperature with 0.4 mM AMPPD or Cl-AMPPD in 0.1 M diethanolamine, 1 mM MgCl2, pH 10.0, containing 1 mg/ml of either polymeric enhancer 1 or enhancer 2 in a Berthold LB952T luminometer. After 5 min of incubation, chemiluminescence intensities were measured as 5 second integrated relative light units (RLU), and the average of the duplicates was calculated and plotted against alkaline phosphatase concentration in the upper graph of Figure 18. The lower graph shows the ratio of the chemiluminescence signal to the background (no enzyme) chemiluminescence signal plotted as a function of enzyme concentration.

EXAMPLE XXIV

The chemiluminescence signal to noise levels was obtained with 2.83 x 10-13 M alkaline phosphatase and various R-substituted adamantyl-1,2-dioxetane phosphates in the presence of various enhancers. All measurements were performed in a Berthold LB952T luminometer at room temperature. First, the background chemiluminescence values (signal in the absence of the enzyme) of each sample were measured (in triplicate). Each sample consisted of 0.2 µmol dioxetane in 0.45 ml 0.1 M diethanolamine, 1 mM MgCl₂, pH 10.0, without or with 1 mg/ml of the indicated enhancer. The test tubes were placed in the luminometer and the luminescence intensity was measured. Thereafter, the tubes were removed from the luminometer and alkaline phosphatase (50 µl containing 1.415 x 10-16 mol) was added to each tube and the luminescence intensity was measured at 5 and 20 minutes after substrate addition. The final concentration of dioxetane in each tube was 0.4 mM. Table 6 shows the ratio of the chemiluminescence signal obtained in the presence of alkaline phosphatase at 5 and 20 minutes to the background signal (obtained in the absence of the enzyme). TABLE 6 COMPARISON OF CHEMILUMINESCENCE SIGNAL TO NOISE VALUES OF VARIOUS R-SUBSTITUTED AMPPD COMPOUNDS

All enhancers were used at a concentration of 1 mg/ml in 0.1 M DEA, pH 10. Alkaline phosphatase concentration = 2.83 x 10⊃min;13 M. Measurements were performed in a Berthold LB952T luminometer.

EXAMPLE XXV

Table 7 provides the detection limits for alkaline phosphatase detection with the R-substituted dioxetanes, determined using the procedures described in Table 5, Example XXII, with the exception of one instrument which was used to obtain the chemiluminescence intensity readings. The luminometer used in these examples was a LabSystems Luminoskan microtiter plate reader. TABLE 7 ALKALINE PHOSPHATASE DETECTION LIMITS WITH CHEMILUMINESCENCE (AT 2X BACKGROUND) 5 MINUTE INCUBATION 20 MINUTES INCUBATION

1) Buffer: 0.1M diethanolamine, 1mM MgCl2 , 0.02% sodium azide, pH 10.0

2) Signal recorder in a Labsystems Luminoskan microplate reader

EXAMPLE XXVI

Table 8 shows the 20 minute incubation values from Table 7 plus measurements made with a Wilj microplate luminometer. TABLE 8 ALKALINE PHOSPHATASE DETECTION LIMITS USING CHEMILUMINESCENCE (AT 2X BACKGROUND) 20 MINUTE INCUBATION, LABSYSTEMS 23 MINUTES INCUBATION, WILJ

EXAMPLE XXVII

The performance of AMPPD and Cl-AMPPD on a nylon membrane was compared as follows: 12.5 picograms of biotinylated pBR322-(35 mer) in 1 μl was spotted on a dry nylon membrane and cross-linked to the membrane by UV light. The membrane was then wetted with 1X SSC buffer, incubated with 0.2% casein, 0.1% Tween 20 in phosphate buffered saline (PBS) for 1 hour at room temperature, washed 4 times with 0.3% Tween 20 in PBS, washed twice in substrate buffer (0.1 M diethanolamine, 1 mM MgCl2, pH 10.0), and incubated in 0.25 mM dioxetane in substrate buffer for 5 minutes. In this way, two membranes were prepared: one incubated with AMPPD and the second with Cl-AMPPD. The membranes were then hermetically wrapped in plastic, attached to the outside of a 13 x 75 mm test tube and placed in a Turner Model 20-E luminometer equipped with a side-mounted photomultiplier detector and thermally equilibrated to 30ºC. The chemiluminescence intensity was then monitored for 20 to 24 hours. As shown in Figure 19, the chemiluminescence signal was plotted as a function of time for both AMPPD and Cl-AMPPD.

EXAMPLE XXVIII

The half-lives of the maximum chemiluminescence intensities obtained with AMPPD and Cl-AMPPD on PVDF and nylon membranes were calculated from the values obtained in Example XXVII for the nylon membrane. For the PVDF membrane, the revised protocol included a second substrate buffer wash followed by membrane incubation for 5 minutes at room temperature in Tropix NitrolockTM reagent and a subsequent wash with substrate buffer prior to dioxetane substrate addition. The half-lives to the maximum chemiluminescence signal were determined from the plot of the logarithm mus (maximum chemiluminescence signal minus signal at any time point) as a function of time and are given in Table 9. TABLE 9 CHEMILUMINESCENCE DETECTION OF BIOTINYLATED DNA ON MEMBRANES - HALF-LIFE TO MAXIMUM CHEMILUMINESCENCE -

* The PVDF membrane was treated with nitrolock prior to addition of the substrate. 12.5 pg of biotinylated pBR322-35mer was detected with Avidix-AP and 0.24 mM dioxetane in 0.1 M DEA, pH 10.0.

EXAMPLE XXIX

Detection of pBR322 plasmid DNA with a biotinylated pBR322 DNA probe using AMPPD and Cl-AMPPD on PVDF and nylon membranes was performed as follows: pBR322 DNA was denatured by boiling, serially diluted samples in 1X SSC buffer (final mass of DNA per slot is given in Fig. 20) were applied to Immobilin PVDF membrane, Pall Biodyne A nylon membrane, and Amersham Hybond N nylon membrane using a Schleicher-Schuell slot blot device. DNA was cross-linked to the Biodyne A and Hybond N membranes using UV light. The Immobilon P membrane was first blocked and then treated with UV. The membranes were then wetted with 1 X SSC buffer, prehybridized in a hybridization buffer (1 M NaCl, 0.2% heparin, 0.5% polyvinylpyrrolidone, 4% sodium dodecyl sulfate, 1 mM ethylenediaminetetraacetic acid, 5% dextran sulfate, 50 mM Tris-HCl, pH 7.5) and then hybridized at 65ºC with 12 ng/ml pBR322 probe (biotinylated using a nick translation method) in the same buffer, washed once at 65ºC in the same buffer minus dextran sulfate, EDTA and heparin, washed twice for 10 minutes with 1 X SSC/1% SDS at 75ºC, washed twice for 15 minutes with 0.1 X SSC/1% SDS at 75ºC washed twice for 5 min in 1X SSC at room temperature, blocked for 1 hour with 0.2% casein, 0.1% Tween 20 in PBS, washed once for 5 min with 0.2% casein in PBS, incubated for 30 min in a 1 to 15,000 dilution of streptavidin-labeled alkaline phosphatase in 0.2% casein/PBS, then washed 6 times with 0.2% casein, Tween 20 in PBS, washed twice in substrate buffer (0.1 M diethanolamine, 1 mM MgCl2, pH 10.0), and then incubated for 5 min in 0.25 mM AMPPD and Cl-AMPPD (two separate membranes) in substrate buffer. Prior to substrate addition, the PVDF membrane was incubated in NitroßlockTM for 5 minutes and washed twice with substrate buffer. The membranes were then wrapped in plastic and exposed to Polaroid type 612 film at the times indicated after substrate additions as shown in Figure 20.

EXAMPLE XXX

Detection of pBR322 plasmid DNA with a biotinylated pBR322 DNA probe using AMPPD, Cl-AMPPD and Br-AMPPD on PVDF, nylon and nitrocellulose membranes was performed using protocols similar to those described in Example XXIX, with an exception in the case of PVDF and nitrocellulose membranes to which the target DNA was fixed by heating the membranes at 80°C for 2 hours. Furthermore, prior to dioxetane addition, both the PVDF and nitrocellulose membranes were treated with Nitroblock. Polaroid Type 612 instant film images of the chemiluminescent signal from the decomposition of various dioxetane phosphates catalyzed by alkaline phosphatase-DNA probe conjugates on various membrane supports are shown in Figure 21.

EXAMPLE XXXI

The kinetic values of alkaline phosphatase-dependent light emission of AMPPD and Cl-AMPPD on nitrocellulose membranes were compared according to the following procedure: pBR322-(35-mer), which had previously been labeled at the 3'-end with biotin-11-dUTP (1 μl, containing 12.5 pg), was spotted onto a dry nitrocellulose membrane. The membrane was then wetted with 1X SSC buffer, incubated with 0.2% casein, 0.1% Tween 20 in phosphate buffered saline for 1 hour at room temperature, washed four times with 0.3% Tween 20 in PBS, washed twice with phosphate buffer (0.1 M diethanolamine, 1 mM MgCl2, pH 10.0), incubated with NitroßlockTM reagent for 5 minutes, washed twice with substrate buffer, and then two pieces of the membrane were incubated for 5 minutes each in 0.25 mM AMPPD and Cl-AMPPD in substrate buffer. The membranes were then tightly packed in plastic, attached to the outside of a 12 x 75 mm test tube, and inserted into a Turner Model 20-E luminometer thermally equilibrated to 30ºC and equipped with a side photomultiplier detector. The chemiluminescence intensity was monitored for 20 to 24 hours. As shown in Figure 22, the chemiluminescence intensity signal was plotted as a function of time for both AMPPD and Cl-AMPPD.

EXAMPLE XXXII

The half-life to maximum chemiluminescence signal intensity for a protein at a membrane was evaluated. IgG detection with alkaline phosphatase conjugated goat anti-mouse antibodies and AMPPD and Cl-AMPPD was determined using the following protocol. 1 μl of 1 μg/ml solution of purified mouse IgG in 20% methanol electrophoresis transfer buffer was spotted onto a dry nitrocellulose and wet PVDF membrane. The membranes were then incubated with 0.1% Tween 20/PBS, blocked for 1 hour in a blocking buffer (0.2% casein, 0.1% Tween 20 in PBS), rinsed with a 1 to 10,000-fold dilution of alkaline phosphatase-conjugated goat anti-mouse antibody in blocking buffer, bloc cation buffer, washed twice for 5 minutes with 0.1% Tween 20-PBS, washed twice for 5 minutes with substrate buffer (0.1 M diethanolamine, 1 mM MgCl2, pH 10.0), divided into two samples and incubated for 5 minutes each with 0.25 mM AMPPD and Cl-AMPPD. The membranes were hermetically packaged in plastic, attached to the outside of a 12 x 75 mm glass tube and inserted into a Turner Model 20E luminometer equipped with a side photomultiplier detector and thermally equilibrated to 30°C. As shown in Table 10, the chemiluminescence intensities were monitored for 8 to 12 hours and the half-life to maximum signal was determined as calculated in Example XXVIII. TABLE 10 CHEMILUMINESCENCE DETECTION OF MOUSE IgG ON MEMBRANES - HALF-LIFE TO MAXIMUM CHEMILUMINESCENCE -

* The PVDF membrane was treated with nitrolock prior to addition of the substrate. 1 ng mouse IgG was detected with goat anti-mouse AP and 0.25 mM dioxetane in 0.1 M DEA, pH 10.0:

EXAMPLE XXXIII

Western blot analysis was performed for detection of human transferrin on a nylon membrane using chemiluminescence. Serial dilutions of purified human transferrin at 0-5,1,2,4,8,16,32,64,128 and 256 nanograms were separated by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to a Pall Biodyne A nylon membrane. Membranes were washed sequentially with phosphate buffered saline (PBS), blocked with 0.3% casein in PBS for 30 minutes, incubated with a 1- to 1000-fold dilution of mouse anti-human transferrin in 0.3% Tween 20/PBS, washed four times for 5 minutes with 0.3% Tween 20/PBS, incubated with a 1- to 10,000-fold dilution of alkaline phosphatase-labeled goat anti-mouse antibody in 0.3% Tween 20/PBS, washed four times for 5 minutes in 0.3% Tween 20/PBS, washed twice with substrate buffer (0.1 M diethanolamine, 1 mM MgCl2, pH 10.0), washed for 5 minutes each in 0.25 mM AMPPD, Cl-AMPPD and Br-AMPPD were incubated in substrate buffer, wrapped in plastic and then exposed to Polaroid Type 612 instant black and white film for 5 minutes. The results are shown in Fig. 30.

EXAMPLE XXXIV

Human transferrin (amounts are indicated in Figure 22) was analyzed by polyacrylamide gel electrophoresis and then transferred to an Immobion PVDF membrane.

Chemiluminescence detection of human transferrin on Immobilon-P PVDF membrane was performed according to the protocol described in Example XXII, except for the following changes: immediately before dioxetane addition, two of the four PVDF membranes were incubated in NitroßlockTM for 5 minutes and then washed twice with substrate buffer. 5 minute exposures for AMPPD and Cl-AMPPD without and with Nitroßlock are shown in Fig. 23.

EXAMPLE XXXV

Chemiluminescence detection of murine interleukin-4 gene using direct alkaline phosphatase labeled oligonucleotide probes with AMPPD, Cl-AMPPD, Br-AMPPD and LumiPhos 530 was performed. A plasmid containing the geneor murine interleukin-4 gene (MIL-4) was serially diluted in 1 X SSC and applied to a dry Biodyne A membrane at 12.8, 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, 0.0063 and 0.00315 nanograms of DNA. The DNA was fixed onto the membrane with UV and then wetted with 1 X SSC, prehybridized with hybridization buffer (7% SDS, 1% casein, 0.25 M Na₂PO₄, pH 7.2 with phosphoric acid) for 30 minutes at 50°C, hybridized with 5 nM alkaline phosphatase-labeled oligonucleotide probe for 2 hours at 50°C and washed as follows: twice for 5 minutes with 2 X SSC, 1% SDS at room temperature, twice for 15 minutes with 1 X SSC, 1% SDS at 50°C, twice for 5 minutes with 1 X SSC, 1% Triton X-100 at room temperature, twice for 5 minutes with 1 X SSC, and twice for 5 minutes in substrate buffer (0.1 M diethanolamine, 1 mM MgCl2, pH 10). Separate samples of the membranes were then incubated for 5 minutes each in 0 to 25 mM AMPPD, Cl-AMPPD, Br-AMPPD and LumiPhos 530 (Boehringer Mannheim, Indianapolis, USA), wrapped in plastic and exposed to Kodak XAR-5 X-ray film for 1 hour after 1 hour of incubation. The results of this experiment are shown in Fig. 24.

EXAMPLE XXXVI

The comparison of the chemiluminescence signal intensities obtained with Cl-AMPPD and AMPPD was examined as shown in Figure 9. Sequencing reactions were performed using M13mp18 DNA with 5'-biotinylated universal primer with BioRad BST polymerase. Two sets of reactions were separated on a 7.67 M urea ion gradient polyacrylamide gel and then transferred to a nylon membrane by passive capillary transfer. The DNA was cross-linked to the membrane by UV irradiation for 5 minutes. The membrane was then inhibited at room temperature in blocking buffer (0.2% casein, 0.1% Tween 20 in PBS) for 30 minutes, incubated for 30 minutes with a 1 to 5000 dilution of streptavidin-labeled alkaline phosphatase in 0.2% casein, PBS, washed for 5 minutes with blocking buffer, washed twice with 0.3% Tween 20 in PBS, and washed for 1 minute with substrate buffer (0.1 M diethanolamine, 1 mM MgCl2, pH 10). The membrane was cut into two pieces and incubated for 5 minutes in 0.25 M dioxetane (one strip each for AMPPD and Cl-AMPPD). The membranes were then sealed in plastic. and exposed to Kodak XAR-5 X-ray film for 7 minutes beginning 5 minutes after substrate addition, as shown in Fig. 25.

EXAMPLE XXXVII

Comparison of DNA band resolution using AMPPD and Cl-AMPPD was performed using sequencing reactions, transfers and chemiluminescence detections were performed under the same conditions as described in Example XXXVI using AMPPD and Cl-AMPPD. The exposure in Figure 26 is a 1-minute exposure 24 hours after substrate addition.

EXAMPLE XXXVIII

Detection of pBR322 plasmid DNA with a biotinylated pBR322 DNA probe using AMPPD, Cl-AMPPD and Br-AMPPD on a nitrocellulose membrane was performed. pBR322 plasmid DNA was serially diluted and spotted at 512, 256, 128, 64; 32, 16, 8, 4, 2 and 1 picogram of DNA onto 3 strips of dry nitrocellulose membrane. The membrane strips were then heated at 80ºC for 2 hours and irradiated with UV light for 5 minutes, wetted with 1 X SSC, prehybridized for 60 minutes in a hybridization buffer (1 M NaCl, 0.2% heparin, 0.5% polyvinylpyrrolidone, 40% sodium dodecyl sulfite, 1 mM ethylenediaminetetraacetic acid, 5% dextran sulfate; 50 mM Tris-HCl, pH 7.5) at 65ºC, hybridized overnight with 15 ng/ml pBR322 probe, biotinylated by nick translation, 5 minutes in hybridization buffer minus heparin, dextran sulfate and EDTA at 65ºC, washed twice for 5 minutes with 1 X SSC/1% SDS at 75ºC, twice during The membranes were then washed for 15 minutes with 0.01 X SSC/1% SDS at 75ºC and washed once for 5 minutes with 1 X SSC at room temperature. The membranes were then further processed for chemiluminescence detection at room temperature by rinsing twice with 0.2% casein in PBS, blocked for 1 hour with blocking buffer (0.20% casein, 0.1% Tween 20 in PBS), washed for 5 minutes with 0; 2% casein in PBS, then washed for 5 minutes with 0.2% casein in PBS, then incubated for 30 minutes with a 1 to 15,000-fold dilution of streptavidin-labeled alkaline phosphatase in 0.2% casein/PBS, washed twice for 5 minutes in nitrolock solution, then washed four times for 5 minutes in 0; 3% Tween-20/PBS, then washed twice for 5 minutes with substrate buffer (0.1 M diethanolamine, 1 mM MgCl2, pH 10), and then one strip was incubated in each of three dioxetane phosphates: AMPPD, Cl-AMPPD and Br-AMPPD at 0.25 M dioxetane concentration.

Time-limited exposures with a Polaroid 612 film after different incubation times are shown in Fig. 27.

EXAMPLE XXXLX

Detection of pBR322 DNA with a biotinylated pBR322 DNA probe with streptavidin-alkaline phosphatase conjugate and chemiluminescent dioxetane phosphates, AMPPD, Cl-AMPPD and Br-AMPPD on a neutral nylon membrane was performed using ing the protocol described in Example XXXVIII, except that the 5 minute nitrolock incubation was omitted. The results are shown in Fig. 28.

EXAMPLE XL

Chemiluminescence detection of human transferrin was performed on an Immobilon PPVDF membrane. Gel electrophoresis, transfer, blocking and detection steps were performed according to the protocol described in Example XXXIV, except that all PVDF membranes were incubated in NitroßlockTM reagent for 5 minutes. Figure 29 shows the comparative results of AMPPD, Cl-AMPPD and Br-AMPPD in the detection of human transferrin on PVDF membranes.

EXAMPLE XLI

The thermal background of each dioxetane, as specified in Table 11, was measured in a solution of 0.4 mM dioxetane in 0.1 M diethanolamine, 1 mM MgCl2, pH 10.0, at 30°C in a Turner Model 20E luminometer (Sunnyvale, CA). The average chemiluminescence intensity of each sample in the absence of enzyme is reported as "nonspecific background" at 0.4 mM in relative luminometer units (TLU) in Table 11.

The half-life of the dioxetane anion, also given in Table 11, was measured as follows: alkaline phosphatase (final concentration) 2.83 x 10-13 M was added to 1 ml of 0.04 mM dioxetane solution (in the same buffer as above) and completely dephosphorylated at 30°C in a Turner Model 20E luminometer. The half-life of the dioxetane anion was then calculated as described in Example XVIII. TABLE 11 HALF-LIFE AND NON-SPECIFIC BACKGROUND EMISSION OF DIOXETANES AT 31.5ºC

* The tube contained 2.83 x 10⊃min;13 M alkaline phosphatase in 0.1 M diethanolamine, 1 mM MgCl₂, 0.02% sodium azide, pH 10.0

The above discussion of the present invention primarily relates to preferred embodiments and practices thereof. It will be apparent to those skilled in the art that further changes and modifications can be readily made in the actual practice of the concepts described herein without changing the scope of the present invention as defined in the claims.

Claims (57)

1. An enzymatically cleavable chemiluminescent 1,2-dioxetane compound capable of generating light energy upon decomposition, which is substantially stable at room temperature before the bond to an enzymatically cleavable labile substituent thereof is intentionally cleaved, represented by the formula: wherein: X and X¹ each represent a substituent in the 5' and 7' positions of the adamant-2'-ylidene substituent, which is individually hydrogen, a hydroxyl group, a halogen substituent, a -O(CH₂)nCH₃ group, where n is 0-19, a hydroxy(lower)alkyl group, a halogen(lower)alkyl group, a phenyl group, a halogenphenyl group, an alkoxyphenyl group, a hydroxyalkoxy group, a cyano group, a carboxyl group or an amido group, where at least one of the substituents X and X¹ has a meaning other than hydrogen, and R₁ and R₂ individually or together represent an organic radical which does not interfere with the generation of light when the dioxetane compound is enzymatically cleaved and satisfies the valence of the 4-carbon atom of the dioxetane compound, with the provisos that when R₁ and R₂ represent individual substituents, the R₂ substituent is an aromatic, heteroaromatic or unsaturated substituent in conjugation with the aromatic ring, and wherein at least one of R₁ and R₂ or R₁ and R₂ together represent a fluorescent chromophoric group substituted with an enzymatically cleavable labile group which luminesces when the enzymatically cleavable labile substituent is removed therefrom by an enzyme. 2. A compound according to claim 1, represented by the formula: wherein Z represents an enzyme-cleavable group, the oxygen on the phenyl ring is ortho, meta or para and Q represents hydrogen, aryl, substituted aryl, aralkyl, heteroaryl having up to 20 carbon atoms, an allyl group, a hydroxy(lower)alkyl group, a lower alkyl-OSiR³ (wherein R³ represents lower alkyl, aryl, alkoxy-C₁₋₆, alkoxyalkyl having 12 or less carbon atoms, hydroxy(lower)alkyl, amino(lower)alkyl), -OR⁴ or -SR⁴ (wherein R⁴ represents alkenyl, substituted (lower)alkenyl, (lower)alkyl or aralkyl having up to 20 carbon atoms), -SO₂R⁵ (wherein R⁵ is methyl, phenyl or NHC₆H₅), substituted or unsubstituted (lower)alkyl, nitro, cyano, halogen, hydroxy, carboxy, trimethylsilyl or a phosphoryloxy group. 3. A compound according to claim 1, characterized in that X is hydroxyl and X¹ is hydrogen. 4. A compound according to claim 1, characterized in that X is chlorine and X¹ is hydrogen. 5. A compound according to claim 1, characterized in that X is bromine and X¹ is hydrogen. 6. A compound according to any one of claims 3 to 5 inclusive, characterized in that R₁ is methoxy. 7. A compound according to claim 6, characterized in that R2 represents a meta-phosphate-substituted phenoxy group. 8. A compound according to claim 7, characterized in that the phosphate substitutent is present as a disodium salt. 9. A compound according to claim 6, characterized in that 1% represents a meta-β-D-galactoside-substituted phenoxy group. 10. A compound according to claim 1, selected from the group consisting of Disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'-hydroxy) tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate, Disodium 3-(4-methoxyspiro[1,2-dioxetane-3, 2'-(5'-chloro) tricyclo[3.3.1.13,7]decane]-4-yl) phenyl phosphate, Disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'-bromo)tricyclo[3.3.1.13.7]decane]-4-yl)phenyl phosphate, syn-Disodium-3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'-hydroxy) tricyclo [3. 3. 1.13.7] decan] -4-yl) phenyl phosphate, syn-disodium-3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13.7]decane]-4-yl)phenyl phosphate, syn-disodium-3-(4-methoxyspiro[1,2-dioxetane.-3,2'-(5'-bromo)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate, anti-disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2' - (5' -hydroxy) tricyclo [3.3.1.13,7]decane] -4-yl) phenyl phosphate, anti-disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro) tricyclo[3.3.1.13.7]decane]-4-yl)phenyl phosphate and anti-disodium 3-(4-methoxyspiro[1, 2-dioxetane-3,2'-(5'-bromo) tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate. 11. Enol ethers of the formula: wherein: X and X¹ each represent a substituent at the 5' and 7' positions of the adamant-2'-ylidene substituent, which individually represent hydrogen, a hydroxyl group, a halogen substituent, an unsubstituted lower alkyl group, a hydroxy(lower)alkyl group, a halogen(lower)alkyl group, a phenyl group, a halophenyl group, an alkoxyphenyl group, a hydroxyalkoxy group, a cyano group, a carboxyl group or an amido group, wherein at least one of the substituents X and X¹ has a meaning other than hydrogen; Y represents a lower alkyl group and Y' is hydrogen, a lower alkyl group, a lower alkenyl group, an aralkyl group with up to 20 carbon atoms atoms, an alkali metal cation, an acyl group having 2 to about 14 carbon atoms inclusive, an alkali metal cation, wherein M independently represents a proton, an alkali metal cation, ammonium, substituted ammonium, quaternary ammonium or an H⁺-pyridinium cation. 12. A compound according to claim 11, selected from the group consisting of: disodium 3-(methoxy-5-hydroxytricyclo[3.3.1.13,7]-dec-2-ylidenemethyl)phenyl phosphate, Disodium 3-(methoxy-5-chlorotricyclo [3. 3.1.13,7]dec-2-ylidenemethyl)phenyl phosphate and Disodium 3-(methoxy-5-bromotricyclo[3.3.1.13,7]dec-2-ylidenemethyl)phenyl phosphate. 13. An assay method using an optically detectable reaction to determine the presence or absence of a particular substance in a sample, characterized in that the optically detectable reaction comprises reaction with an enzyme of the enzymatically cleavable chemiluminescent 1,2-dioxetane compound as defined in claim 1. 14. Assay method according to claim 13, characterized in that X is chlorine and X¹ is hydrogen. 15. Assay method according to claim 13, characterized in that the 1,2-dioxetane compound Disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro) tricyclo[3. 3. 1.13,7]decane] -4-yl) phenyl phosphate. 16. An assay method according to claim 13, comprising the following steps: (i) treating a sample suspected of containing a detectable substance with a buffered solution containing an enzyme bound to a substance having a specific affinity for the detectable substance, (ii) incubating the resulting solution to allow the detectable substance to bind to the specific affinity moiety of the specific affinity enzyme compound, (iii) washing off the excess of the specific affinity enzyme compounds, (iv) adding the enzymatically cleavable chemiluminescent 1,2-dioxetane compound as defined in claim 1 containing a group cleavable by the enzyme portion of the specific affinity enzyme compound, (v) detecting the luminescence obtained as an indication of the presence of the detectable substance in the sample and measuring the luminescence to determine the concentration of the substance. 17. Assay method according to claim 16, characterized in that the detectable substance and the substance having a specific affinity for the detectable substance is selected from antigens or antibodies. 18. Assay method according to claim 17, characterized in that the detectable substance is a nucleic acid and that the substance which has a specific affinity for the detectable substance is a probe which can bind to the whole or a part of the nucleic acid. 19. Assay method according to claim 18, characterized in that the probe is a labeled oligonucleotide that is complementary to the nucleic acid. 20. Assay method according to claim 19, characterized in that the DNA, the RNA or the fragment thereof is formed according to a sequencing protocol. 21. An assay method according to claim 20, characterized in that it further comprises the steps of (a) treating the DNA, RNA or fragment thereof with a labeled complementary oligonucleotide probe to form a hybridization pair, (b) treating the hybridized pair with a molecule capable of binding strongly to the label of the oligonucleotide covalently conjugated to an enzyme capable of cleaving an enzymatically cleavable 1,2-dioxetane to release light energy, (c) adding a 1,2-dioxetane substrate and (d) detecting the light formed. 22. Assay method according to claim 21, characterized in that the oligonucleotide label is biotin or a biotin derivative. 23. Assay method according to claim 21, characterized in that the molecule which can strongly interact with the label of the oligonucleotide is avidin or streptavidin. 24. Assay method according to claim 19, characterized in that the oligonucleotide probe is covalently labeled with an enzyme that can decompose the 1,2-dioxetane by emitting light energy. 25. Assay method according to claim 19, characterized in that the label of the oligonucleotide probe comprises a covalently bound antigen which is immunochemically bound to an antibody-enzyme conjugate, the antibody is directed against the antigen and the enzyme can decompose the 1,2-dioxetane compound with the emission of light energy. 26. Assay method according to one of claims 24 or 25, characterized in that the enzyme is an acid or alkaline phosphatase, R₁ is methoxy and R₂ is a meta-phosphate-substituted phenoxy group. 27. Assay method according to one of claims 17, 19, 24 or 25, characterized in that the binding of the probe to the nucleic acid is carried out on a nylon membrane. 28. Assay method according to one of claims 21 to 23 inclusive, characterized in that that the hybridization between the DNA, the RNA or a fragment thereof and the labeled oligonucleotide probe is carried out on a nylon membrane. 29. Assay method according to claim 13, carried out using a solid matrix, characterized in that the non-specific binding to the Matrix is blocked by pretreatment of the matrix with a polymeric quaternary ammonium salt. 30. Assay method according to claim 29, characterized in that the assay comprises pretreating a nitrocellulose membrane with BDMQ and TBQ. 31. Assay method according to claim 29, characterized in that the assay comprises pretreating the PVD membrane with BDMQ and TBQ. 32. An assay method according to claim 13, characterized in that it is further carried out in the presence of a water-soluble enhancing substance which increases the specific light energy production above the value produced in its absence. 33. Assay method according to claim 32, characterized in that the enhancing substance is a polymeric quaternary ammonium salt. 34. Assay method according to claim 33, characterized in that the polymeric quaternary ammonium salt is poly(vinylbenzyltrimethylammonium chloride), poly[vinylbenzyl(benzyldimethylammonium chloride)] or poly[vinyl(benzyltributylammonium chloride)]. 35. Assay method according to claim 32, characterized in that the enhancing substance comprises a positively charged polymeric quaternary ammonium salt and a fluorescein which forms a ternary complex with the negatively charged product of the 1,2-dioxetane compound formed after enzyme-catalyzed decomposition of the 1,2-dioxetane compound, whereby energy transfer occurs between the negatively charged product and fluorescein and light energy is emitted from the fluorescein. 36. Assay method according to claim 35, characterized in that the polymeric quaternary ammonium salt is poly(vinylbenzyltrimethylammonium chloride), poly[vinylbenzyl(benzyldimethylammonium chloride)] or poly[vinyl(benzyltributylammonium chloride)]. 37. A kit for detecting a first substance in a sample comprising the enzymatically cleavable chemiluminescent 1,2-dioxetane compound as claimed in claim 1 and an enzyme capable of cleaving the enzymatically cleavable group of the 1,2-dioxetane compound. 38. Kit according to claim 37, characterized in that R₁ is methoxy; R₂ is a metaphosphate-substituted phenoxy group and the enzyme is an acid or alkaline phosphatase. 39. A kit for detecting a nucleic acid or a fragment thereof in a sample by hybridizing the nucleic acid or fragment thereof to a complementary labeled oligonucleotide probe, comprising the enzymatically cleavable chemiluminescent 1,2-dioxetane compound of claim 1, an oligonucleotide probe covalently labeled with an enzyme, and a nylon membrane on which the nucleic acid-oligonucleotide probe hybridization is carried out. 40. Kit according to claim 39, characterized in that R₁ is methoxy, R₂ is a metaphosphate-substituted phenoxy group and that the enzyme is an acid or alkaline phosphatase. 41. Kit for the detection of a nucleic acid or a fragment thereof as defined in claim 39, characterized in that the complementary oligo gonucleotide probe covalently labeled with biotin or a biotin derivative, avidin or streptavidin. 42. Kit according to claim 41, characterized in that R₁ is methoxy, R₂ is a metaphosphate-substituted phenoxy group and the enzyme is an acid or alkaline phosphatase. 43. A kit for detecting a nucleic acid or a fragment thereof in a sample by hybridizing the nucleic acid or fragment thereof to a complementary labeled oligonucleotide probe comprising an enzymatically cleavable chemiluminescent 1,2-dioxetane compound, a complementary oligonucleotide probe covalently labeled with an antigen, an antibody directed against the antigen covalently bound to an enzyme capable of decomposing the 1,2-dioxetane compound with the emission of light energy, and a nylon membrane on which the nucleic acid or fragment thereof is hybridized with the oligonucleotide probe. 44. Kit according to claim 43, characterized in that the enzyme is an acid or alkaline phosphatase, R₁ is methoxy and R₂ is a metaphosphate-substituted phenoxy group. 45. A kit for detecting a protein in a sample, comprising an enzymatically cleavable chemiluminescent 1,2-dioxetane compound as defined in claim 1, an antibody directed against the protein covalently linked to an enzyme capable of decomposing the 1,2-dioxetane compound with the emission of light energy, and a membrane on which the protein-antibody binding is carried out. 46. Kit according to claim 45, characterized in that the membrane is nylon, a nitrocellulose membrane or a PVDF membrane. 47. Kit according to claim 45, characterized in that R₁ is methoxy, R₂ is a metaphosphate-substituted phenoxy group and the enzyme is an acid or alkaline phosphatase. 48. A kit for detecting a protein in a sample as defined in claim 45, further comprising a second antibody directed against the first antibody covalently linked to an enzyme capable of degrading the 1,2-dioxetane compound. 49. Kit according to claim 48, characterized in that R₁ is methoxy, R₂ is a metaphosphate-substituted phenoxy group and the enzyme is acid or alkaline phosphatase. 50. A kit according to any one of claims 37, 39, 41, 43, 45 or 48, further comprising a water-soluble enhancing substance that increases the specific light energy production above the value produced in its absence. 51. Kit according to claim 50, characterized in that the reinforcing substance is a polymeric quaternary ammonium salt. 52. Kit according to claim 50, characterized in that the polymeric quaternary ammonium salt is poly(vinylbenzyltrimethylammonium chloride), poly[vinylbenzyl(benzyldimethylammonium chloride)] or poly[vinylbenzyl(tributylammonium chloride)]. 53. Kit according to claim 50, characterized in that the enhancement substance comprises a positively charged polymeric quaternary ammonium salt and a fluorescein which can form a ternary complex with the negatively charged product of the 1,2-dioxetane compound formed after the enzymatically catalyzed decomposition of the 1,2-dioxetane compound, whereby energy transfer takes place between the negatively charged product and the fluorescein and light energy is emitted from the fluorescein. 54. Kit according to claim 53, characterized in that the polymeric quaternary ammonium salt is poly(vinylbenzyltrimethylammonium chloride), poly[vinylbenzyl(benzyldimethylammonium chloride)] or poly[vinylbenzyl(tributylammonium chloride)]. 55. A method for detecting an enzyme in a sample, comprising the steps of: (a) providing an enzymatically cleavable chemiluminescent 1,2-dioxetane compound as claimed in claim 1, (b) treating the 1,2-dioxetane compound with the sample containing the enzyme, whereby the enzyme cleaves the enzymatically cleavable labile substituent from the 1,2-dioxetane compound to form a negatively charged substituent bonded to the 1,2-dioxetane compound, whereby the negatively charged substituent causes the 1,2-dioxetane compound to decompose to form a luminescent substance containing the fluorescent chromophoric group, and (c) Detection of the luminescent substance as an indication of the presence of the enzyme. 56. Process according to claim 55, characterized in that R₁ is methoxy, R₂ is a meta phosphate-substituted phenoxy group and the enzyme is an acid or alkaline phosphatase. 57. A process according to claim 55, characterized in that R₁ is methoxy and R₂ is a meta-β-D-galactoside-substituted phenoxy group and the enzyme is a galactosidase.